PubMed Health⌕ Search

Biomedical subjects

M Sinensky

Publications and source records attributed to M Sinensky.

At least 37 records · Page 2Linked to original sources
PubMed1992

The prenylation of proteins.

The prenylated proteins represent a newly discovered class of post-translationally modified proteins. The known prenylated proteins include the oncogene product p21ras and other low molecular weight GTP-binding proteins, the nuclear lamins, and the gamma subunit of the heterotrimeric G proteins. The modification involves the covalent attachment of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety in a thioether linkage to carboxyl terminal cysteine. The nature of the attached substituent is dependent on specific sequence information in the carboxyl terminus of the protein. In addition, prenylation entrains other posttranslational modifications forming a reaction pathway. In this article, we review our current understanding of the biochemical reactions involved in prenylation and discuss the possible role of this modification in the control of cellular functions such as protein maturation and cell growth.

Amino Acid Sequence↗
PubMed1992

p23rab2, a ras-like GTPase with a -GGGCC C-terminus, is isoprenylated but not detectably carboxymethylated in NIH3T3 cells.

With the development of a specific anti-rab2 antiserum, p23rab2, a ras-like GTPase with a -GGGCC C-terminus, has been localized mainly to the particulate (P100) fraction in NIH3T3 cells, although a small amount of this protein also appears in the soluble fraction. The endogenous p23rab2 is isoprenylated in intact cells, and recombinant murine p23rab2 is also isoprenylated in an in vitro system using reticulocyte lysate. Both the cytosolic and membrane-bound forms of p23rab2 are isoprenylated in intact cells. Recombinant p23rab2 is specifically geranylgeranylated at one or both of the C-terminal cysteine residues in the in vitro system. Blocking isoprenoid synthesis with lovastatin results in an accumulation of a totally cytosolic unisoprenylated form, indicating that isoprenylation is a prerequisite for membrane association of p23rab2. Surprisingly, unlike p21ras and p25rab3A, no carboxymethylation of p23rab2 is detectable in either the soluble or particulate fractions.

3T3 Cells↗
PubMed1991

Isoprenoid modification of rab proteins terminating in CC or CXC motifs.

Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.

Amino Acid Sequence↗
PubMed1990

Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis.

It has been reported that when 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors are utilized for treatment of hypercholesterolemia, as much as 50% inhibition of whole body cholesterol biosynthesis is observed. As general inhibitors of isoprenoid biosynthesis, these compounds can also inhibit the synthesis of the substituents of isoprenylated proteins. For two mammalian proteins (p21ras and lamin A), it has been demonstrated that such inhibition of biosynthesis of the isoprenoid substituent blocks proteolytic maturation of these proteins. It has been argued that advantage may be taken of this phenomenon to block the synthesis of p21ras in malignancies. It is also possible that treatment of hypercholesterolemia with lovastatin might produce problematic inhibition of protein processing dependent upon isoprenylation. In this report, we compare the concentration dependence of inhibition of isoprenylation dependent protein processing and sterol biosynthesis. Effects of partial inhibition of isoprenylated protein processing on whole cells can be sensitively assessed by visualization of lamina structure through indirect immunofluorescence. Our results indicate that the degree of inhibition of p21ras and prelamin A maturation by lovastatin is identical. Thus, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors are unlikely to be useful as anti-malignancy drugs. However, the conditions of lovastatin treatment which produce 50% inhibition of sterol biosynthesis analogous to pharmacological conditions, produce no observable effects on isoprenylated protein maturation.

Animals↗
PubMed1990

Defective elongation of fatty acids in a recessive 25-hydroxycholesterol-resistant mutant cell line.

The Chinese hamster ovary recessive mutant, crB, has been selected for its resistance to the cytotoxic effects of 25-hydroxycholesterol in sterol-free media (Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell. Physiol. 113, 314-319). Growth of crB in a chemically defined lipid-poor medium is very slow and is enhanced by a mixture of saturated and unsaturated fatty acids. Incorporation of [3H]acetate into total fatty acids is 4-fold lower in crB compared to that in parental Chinese hamster ovary K1 and in contrast to the wild-type cells, crB cells are unable to synthesize either stearate or oleate. In addition, crB cells can not elongate exogenous palmitate, while they are capable of desaturating exogenous stearate. The mutant cells are also pleiotropically defective in the regulation of mRNA levels for the enzymes of cholesterol biosynthesis. 25-Hydroxycholesterol is a poor regulator of the synthesis and degradation of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase in crB in comparison to the wild-type Chinese hamster ovary K1 cells. The defect in the elongation of fatty acids is reversed in revertants of crB selected for their ability to grow in lipid-poor medium. Such revertants exhibit normal regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by 25-hydroxycholesterol. Regulation of reductase activity in crB cells can also be restored by supplementing the culture medium with a mixture of fatty acids that restores normal growth rate. The defective regulation of reductase in crB does not appear to be due to nonspecific adverse effects of fatty acid starvation nor is it due to any gross change in the fatty acid composition of cellular phospholipids. These results strongly suggest a direct relationship between the fatty acid auxotrophy of crB and defective regulation of the enzymes of cholesterol biosynthesis.

Acetates↗
PubMed1990

Inhibition of isoprenoid biosynthesis and the post-translational modification of pro-p21.

It has recently been reported that a precursor of p21ras (pro-p21ras) becomes modified by a metabolite of mevalonic acid prior to conversion to mature p21ras. We have examined the effect of blocking isoprenoid biosynthesis on this process. Fluoromevalonate, which inhibits the conversion of pyrophosphomevalonate to isopentenyl pyrophosphate, blocks the incorporation of radioactive mevalonate into pro-p21ras, demonstrating the mevalonate must be converted to an isoprenoid prior to such incorporation. Starvation of CHO-K1 cells for mevalonic acid by treatment with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, or mevalonate deprivation in a mevalonate auxotroph defective in HMG-CoA synthase activity results in the accumulation of pro-p21ras. The precursor, accumulated due to either of these treatments, is converted through an intermediate form to the mature p21ras by incubation of cells with mevalonate. Incubation of cells with 25-hydroxycholesterol, the pleiotropic transcriptional down-regulator of cholesterol biosynthesis does not, however, result in the accumulation of pro-p21ras. This result indicates that in contrast to the regulation of cholesterol biosynthesis in mammalian cells, important regulatory control other than at the level of HMG-CoA reductase is involved in the isoprenoid biosynthesis required for protein isoprenylation.

Animals↗
PubMed1990

Isoprenylation is required for the processing of the lamin A precursor.

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. Moreover, inhibition of prelamin A processing by mevinolin treatment is rapidly reversed by the addition of exogenous mevalonate. Processing of prelamin A is, therefore, dependent on isoprenoid metabolism. Analysis of the conversion of prelamin A to lamin A by two independent methods, immunoprecipitation and two-dimensional nonequilibrium pH gel electrophoresis, demonstrates that a precursor-product relationship exists between prelamin A and lamin A. Analysis of R,S-[5-3H(N)]mevalonate-labeled cells shows that the rate of turnover of the isoprenoid group from prelamin A is comparable to the rate of conversion of prelamin A to lamin A. These results suggest that during the proteolytic maturation of prelamin A, the isoprenylated moiety is lost. A significant difference between prelamin A processing, and that of p21ras and the B-type lamins that undergo isoprenylation-dependent proteolytic maturation, is that the mature form of lamin A is no longer isoprenylated.

Amino Acid Sequence↗
PubMed1989

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity.

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.

Acetyl-CoA C-Acetyltransferase↗
PubMed1989

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover.

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.

Alkaline Phosphatase↗
PubMed1988

Somatic cell genetics and the study of cholesterol metabolism.

The regulation of cholesterol biosynthesis by extracellular cholesterol occurs both in whole animal tissue and in permanent somatic cell lines in culture. Permanent mammalian cells lines, under optimized growth conditions, are easily manipulated both biochemically and genetically. The Chinese hamster ovary cell line (CHO-K1) is the most widely used cell line for genetic studies. CHO-K1 is a pseudo-diploid mammalian cell exhibiting a short doubling time and a relatively high plating efficiency. Somatic cell mutants can be generated through mutagenesis and also by drug adaptation. Following mutagenesis, auxotrophs may be isolated either by selection or by screening. Most selection procedures for mutants of cholesterol metabolism must be done in serum depleted of cholesterol which requires the endogenous biosynthetic pathway to be intact. Mutants failing to produce cholesterol do not replicate their DNA and exhibit reduced concentrations of cholesterol in their membranes. BUdR and polyene antibiotics have both been used to select against the wild-type cells which incorporate these compounds and are killed, allowing the survival of the mutant cells. Both mevalonate and cholesterol auxotrophs have been isolated with the BUdR technique and have proven useful for elucidation of the early steps in cholesterol biosynthesis, particularly for the ratelimiting enzyme HMG-CoA reductase. Somatic cell fusion of a mutant and wild-type cell followed by chromosomal segregation, routinely used to map human genes, has also been used to map the human gene for HMG-CoA synthase. Such hybrids also provide valuable information on the dominance or recessivity of a specific lesion. DNA-mediated gene transfer into somatic cell mutants allows the selection of DNA sequences which complement the mutation, and is also useful for analysis of regions of regulatory significance. Mutants, resistant to the regulatory effects of oxygenated sterols, can be isolated following mutagenesis. Mutants of this type vary the lipid content of their membranes in response to cholesterol concentration in the medium. All such mutants tested exhibit a pleiotropic regulatory effect on more than one enzyme in the cholesterol biosynthetic pathway. Adaptation to drugs such as compactin and mevinolin, which inhibit HMG-CoA reductase, have been used to produce mutants which overexpress enzymes in the pathway. These amplified cells are useful sources of specific mRNAs for construction of cDNA libraries and gene isolation. Structure-function relationships of membrane sterols can be studied in cholesterol auxotrophs where changes in acyl-chain ordering can be manipulated by exogenous sterols in the medium.

Animals↗
PubMed1988

Further characterization of a somatic cell mutant defective in regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Two enzymes of mammalian cellular mevalonate biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, have been shown to be regulated by exogenous sterols. It has been demonstrated that these enzymes are regulated, at least in part, by transcriptional control of their synthesis. We have previously described a somatic cell mutant (CR1) of the CHO-K1 cell line that is defective in regulation of the activity of these enzymes in response to exogenous sterols. In this report, we demonstrate that this mutant is defective in regulation of the mRNA levels for HMG-CoA reductase and HMG-CoA synthase by 25-hydroxycholesterol and mevinolin. In the case of HMG-CoA reductase, this loss of apparent transcriptional control is not accompanied by a comparable loss in regulation of synthesis of this enzyme. This observation is consistent with prior studies suggesting that HMG-CoA reductase can be regulated translationally. We also show that CR1 cells exhibit a constitutively rapid rate of degradation of HMG-CoA reductase.

Animals↗
PubMed1988

Incorporation of a product of mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei.

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, non-equilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified.

Animals↗
PubMed1987

Characterization of HMG CoA synthase activity of rat liver and CHO-K1 cells.

This report describes the characterization and partial purification of rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase activity. A preliminary characterization of Chinese hamster ovary (CHO) cell HMG CoA synthase activity is also presented. Ion-exchange chromatography of ammonium sulfate precipitates of rat liver cytosol indicate the existence of two isoenzymes of HMG CoA synthase. These isoenzymes are physically, catalytically, and immunologically distinct. One of these isoenzymes, peak 1, resembles mitochondrial HMG-CoA synthase activity as evidenced by similarities in elution upon ion-exchange chromatography, inhibition by MgCl2, and cross reactivity with an antibody prepared against the mitochondrial enzyme. As peak 1 activity is unstable, further purification studies were performed on peak 2 activity. Peak 2 can be further resolved into two activities (peaks 2A and 2B) by gel filtration. In contrast, CHO-K1 cells (a permanent fibroblast line) possess only peak 2 type HMG CoA synthase activity.

Animals↗
PubMed1987

Requirement for 24(S),25-epoxycholesterol for the viability of cultured fibroblasts.

Previous studies on a somatic cell mutant auxotrophic for mevalonate (Mev-1) have shown that these cells rapidly lose viability when deprived of mevalonic acid in culture medium supplemented with serum cholesterol. Testing of all known end products of mevalonate metabolism in cultured mammalian cells has been conducted to determine the basis for this mevalonate requirement. It has been found that the recently discovered mevalonate metabolite 24(S),25-epoxycholesterol produces a partial restoration of viability of Mev-1 cells starved for mevalonate, whereas other structurally similar oxysterols do not. It appears that 24(S),25-epoxycholesterol has a specific, vital cellular function in CHO-K1 cells.

Animals↗
PubMed1987

Endogenous sterol synthesis is not required for regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by low density lipoprotein.

It has been proposed that an endogenously synthesized oxysterol mediates the regulation of cholesterol biosynthesis by low density lipoprotein in cultured mammalian cells. Studies in this report demonstrate that under conditions in which squalene conversion to sterols is blocked either by inhibition of squalene cyclization or lanosterol demethylation, or both, low density lipoprotein regulates 3-hydroxy-3-methylglutaryl coenzyme A reductase normally. These observations rule out the hypotheses that either an endogenously synthesized oxygenated cholesterol biosynthetic intermediate or epoxysterol is required to mediate the inhibition of this enzyme by low density lipoprotein.

Cells, Cultured↗
PubMed1986

Localization of the gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthase to human chromosome 5.

A series of hybrids between primary human cells and a Chinese hamster somatic cell mutant (Mev-1), defective in expression of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating, EC 4.1.3.5], has been prepared that complements the mutant defect. A technique based on differential sensitivity of this enzyme activity to inhibition by magnesium ion is described that allows the discrimination of expression of human and hamster HMG-CoA synthase in these hybrids. The results indicate a structural gene defect in expression of HMG-CoA synthase activity in Mev-1 cells. Segregation of human chromosomes that do not possess the complementing marker have allowed the assignment of human HMG-CoA synthase activity to chromosome 5. This is the second demonstrably transcriptionally regulated enzyme of cholesterologenesis to be assigned to chromosome 5, the other being HMG-CoA reductase.

Chromosome Mapping↗