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Biomedical subjects

M Sinensky

Publications and source records attributed to M Sinensky.

At least 19 recordsLinked to original sources
PubMed2000

Functional aspects of polyisoprenoid protein substituents: roles in protein-protein interaction and trafficking.

There are now numerous examples of post-translational modification with geranylgeranyl or farnesyl substituents. Once thought of as solely a mechanism for association of proteins with membranes, other functional aspects of protein prenylation have come to be appreciated. Although, in almost all instances, such proteins are membrane associated, they are often found to also engage in protein-protein interactions. In some instances, such interactions are critical aspects of prenylated protein trafficking. In this review, the role of prenylation in mediating protein-protein interactions will be considered. The hypothesis will be developed that such interactions occur through recognition of the prenyl group and a second domain, on the prenylated protein, by a heterodimeric protein partner.

Animals↗
PubMed2000

Recent advances in the study of prenylated proteins.

Post-translational modification of proteins with isoprenoids was first recognized as a general phenomenon in 1984. In recent years, our understanding, including mechanistic studies, of the enzymatic reactions associated with these modifications and their physiological functions has increased dramatically. Of particular functional interest is the role of prenylation in facilitating protein-protein interactions and membrane-associated protein trafficking. The loss of proper localization of Ras proteins when their farnesylation is inhibited has also permitted a new target for anti-malignancy pharmaceuticals. Recent advances in the enzymology and function of protein prenylation are reviewed in this article.

Alkyl and Aryl Transferases↗
PubMed1999

Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A.

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease.

Acrylic Resins↗
PubMed1999

Concepts in Ras-directed therapy.

Ras proteins are key transducers of growth signals regulated by cell surface receptors. They are anchored to the inner surface of the cell membrane where receptor-mediated signalling induces Ras activation (GDP/GTP exchange) and inactivation (stimulation of Ras GTPase activity). Ras-GTP in turn activates a multitude of signalling cascades controlling cell growth and differentiation. Aberrant Ras function (mostly constitutive activation) contributes to the development of many types of neoplastic human diseases. Activating mutations in ras genes, leading to the expression of Ras proteins insensitive to Ras-GTPase activating proteins, are found in as many as 30% of all human tumours. This suggests that Ras is an appropriate target for drug design. Remarkable improvements in the understanding of post-translational modifications in Ras that promote Ras-membrane anchorage, in the mechanisms of activation and inactivation of Ras, and in the interactions of Ras with a plethora of effector molecules have led to the development of new concepts for Ras-directed therapy. The most advanced approach has been that of farnesyltransferase inhibitors (FTIs) designed to inhibit the farnesylation of Ras required for membrane anchorage and transforming activity. FTIs now in clinical trials have been extensively reviewed. Here we review the progress in the development of FTIs and in the development of other promising concepts for Ras-directed therapy. These include compounds such as S-farnesylthiosalicylic acid (FTS), which disrupt the proper anchorage of Ras with the cell membrane and inhibit human tumour growth in animal models, and compounds that interfere with interactions of Ras with its downstream effectors. We conclude with a description of a recently described novel drug concept that could restore the defective GTPase activity of oncogenic Ras and with the interesting results of reovirus-induced tumour regression observed in animal models of human tumours containing an intact Ras signalling pathway.

Journal Article↗
PubMed1998

Oleate potentiates oxysterol inhibition of transcription from sterol regulatory element-1-regulated promoters and maturation of sterol regulatory element-binding proteins.

Activation of genes containing SRE-1 (sterol regulatory element 1) sequences is known to be under the regulation of sterols through modulation of the proteolytic maturation of SREBPs (SRE-1-binding proteins). Previous work has demonstrated SREBP-mediated transcriptional activation of genes encoding enzymes of sterol and fatty acid biosynthesis. Because synthesis of both sterols and C18 fatty acids are required for cell growth, in the absence of exogenous supplements of these lipids, we examined the hypothesis that fatty acid can also be regulatory in SREBP maturation. Our data indicate that C18 fatty acids can potentiate the biological activities of a typical, regulatory sterol: 25-hydroxycholesterol. Inhibition of C18 fatty acid synthesis in cells cultured in serum-free medium renders them resistant to killing by 25-hydroxycholesterol. Repression of expression of reporter constructs driven by promoters bearing SRE-1 element(s) by 25-hydroxycholesterol is increased by C18 fatty acid supplementation. C18 fatty acids also increase the inhibitory effect of 25-hydroxycholesterol on proteolytic maturation and nuclear localization of SREBPs. Furthermore, we also show that C18 fatty acid supplementation can enhance the inhibitory effect of 25-hydroxycholesterol on sterol and fatty acid biosynthesis. These results demonstrate that maximal down-regulation of SREBP maturation and the consequent repression of SRE-1 promoters occurs in response to both a regulatory sterol and fatty acid.

Alkynes↗
PubMed1998

Evidence for a high affinity, saturable, prenylation-dependent p21Ha-ras binding site in plasma membranes.

Oncogenic p21ras proteins can only exert their stimulation of cellular proliferation when plasma membrane-associated. This membrane association has an absolute requirement for post-translational modification with isoprenoids. The mechanism by which isoprenoids participate in the specific association of p21ras with plasma membranes is the subject of this report. We present in vitro evidence for a plasma membrane binding protein for p21(ras) that can recognize the isoprenoid substituent and, therefore, may facilitate the localization of p21ras.

3T3 Cells↗
PubMed1997

Regulation of prelamin A endoprotease activity by prelamin A.

The maturation of lamin A is completed by the endoproteolytic cleavage of its farnesylated precursor protein, prelamin A. In the absence of this cleavage, prelamin A can neither give rise to lamin A nor assemble into the nuclear lamina. We call the enzyme which catalyzes this endoproteolytic step the 'prelamin A endoprotease'. In this study, we begin characterization of the regulation of prelamin A endoprotease. In particular, we address the question as to whether prelamin A endoprotease activity is constitutive in cells or responds to expression of prelamin A. To do this, we compared the activity of this novel endoprotease in cells which express prelamin A with those that do not. Our data shows that the enzymatic activity of prelamin A endoprotease is enhanced by the expression of prelamin A.

Chromatography, Thin Layer↗
PubMed1997

In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.

The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S-farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr657 and Leu658. Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.

Amino Acid Sequence↗
PubMed1997

Strongyloides stercoralis: identification of antigens in natural human infections from endemic areas of the United States.

Using Western-blot analysis, we identified eight immunodominant antigens (apparent molecular weights 96, 86, 75, 56, 41, 32, 28, and 26 kDa) of Strongyloides stercoralis in natural human infections. For this study, 78 individual serum samples were obtained from S. stercoralis-infected patients residing in endemic areas of the United States. Poly A+ RNA was translated in vitro in the rabbit-reticulocyte lysate system. The newly synthesized translation products were immuno-precipitated with S. stercoralis human infection sera. All eight of the identified antigens were detected in the immunoprecipitates. The potential of these antigens as targets for immunodiagnosis is also discussed.

Animals↗
PubMed1995

The farnesyl protein transferase inhibitor BZA-5B blocks farnesylation of nuclear lamins and p21ras but does not affect their function or localization.

BZA-5B is a peptidomimetic inhibitor of protein farnesylation in mammalian cells. We have examined the specificity of this compound toward inhibition of farnesylation of p21ras and the nuclear lamin proteins, prelamin A and lamin B. We have also used the Raney nickel cleavage technique in conjunction with radio-gas liquid chromatography to assess the ability of this compound to block total protein farnesylation. These studies show that BZA-5B blocks farnesylation of the lamin proteins with an IC50 comparable to that seen for p21ras. At a concentration in excess of 25 microM, BZA-5B inhibits all protein farnesylation in CHO-K1 cells below the limits of detection. Furthermore, we found that after a 2-day exposure to high concentrations of BZA-5B, CHO-K1 cell lines exhibit no loss in sensitivity to inhibition of prenylation by this compound. Yet, despite the potent and general inhibition of protein farnesylation, BZA-5B does not interfere with a variety of cellular functions expected to be farnesylation dependent, including cell growth and viability, assembly of the nuclear lamina, membrane association of p21ras, and p21ras-dependent differentiation of PC-12 cells in response to treatment with nerve growth factor. The maintenance of farnesylation-dependent events in the presence of BZA-5B stands in marked contrast to the inhibition of the oncogenic ras-mediated transformed phenotype that has been observed with this compound and other farnesyl protein transferase inhibitors. This specificity for inhibition of ras transformation by BZA-5B is quite encouraging to its eventual development as an antimalignancy pharmaceutical.

Adrenal Gland Neoplasms↗
PubMed1994

An antibody which specifically recognizes prelamin A but not mature lamin A: application to detection of blocks in farnesylation-dependent protein processing.

A polyclonal antibody [anti-prelamin A antibody (alpha-PA)] has been obtained against the peptide LLGNSSPRTQSPQN which is proteolytically removed during the farnesylation-dependent processing of prelamin A to mature lamin A. We tested the ability of this antibody to detect inhibition of farnesylation-dependent protein processing of prelamin A. The alpha-PA antibody was shown to immunoprecipitate prelamin A from lovastatin-treated HeLa cells but not mature lamin A from untreated cells. Further studies were performed after antigen-affinity chromatographic purification of the antibody. Western blotting of lovastatin-treated HeLa cell extract demonstrated that the purified alpha-PA antibody recognizes prelamin A. Furthermore, this signal could be competed away by incubation with the peptide. Indirect immunofluorescence helped detect nuclear accumulation of the antigen in response to treatment of HeLa cells with lovastatin or in Chinese hamster ovary K1 cells transiently transfected with a prelamin A mutant blocked in farnesylation. This antibody should be useful for screening compounds that may block any of the three common steps in the farnesylation-dependent processing of proteins (farnesylation, endoproteolysis, and carboxymethylation) since it appears that prelamin A undergoes all of these reactions prior to removal of the antigenic peptide. Inhibitors of these reactions have been proposed as potential anticancer drugs, since they would be expected to block the biological activity of oncogenic p21ras proteins. Since such screening would be performed most efficiently by enzyme-linked immunosorbent assays, we can detect the accumulation of prelamin A after treatment with lovastatin by performing this procedure as well. Application of alpha-PA in an enzyme-linked immunosorbent assay, which demonstrates the activity of a peptidomimetic farnesyltransferase inhibitor, supports the use of this antibody in large scale screening for inhibitors of farnesylation-dependent protein processing.

Amino Acid Sequence↗
PubMed1994

The processing pathway of prelamin A.

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins--the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dexamethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and carboxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus.

Amino Acid Sequence↗
PubMed1994

Expression of prelamin A but not mature lamin A confers sensitivity of DNA biosynthesis to lovastatin on F9 teratocarcinoma cells.

The role of inhibition of prelamin A processing in the inhibition of DNA synthesis by lovastatin was examined by expressing prelamin A in F9 teratocarcinoma cells. These cells, normally lacking expression of the A/C lamins, were transfected with constructs expressing either prelamin A or mature lamin A and the effect of lovastatin on DNA biosynthesis was assessed. It was found that expression of prelamin A specifically conferred sensitivity to inhibition of DNA biosynthesis by lovastatin on F9 cells.

Animals↗
PubMed1992

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

3T3 Cells↗
PubMed1992

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 24(S),25-oxidolanosterol.

We have examined the mechanism of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 24(S),25-oxidolanosterol, a C30-sterol naturally occurring in mammalian tissues. In the absence of enzymatic demethylation to the C27-sterol, 24(S),25-epoxycholesterol, oxidolanosterol is shown to be a post-transcriptional regulator of reductase synthesis in both primary rat hepatocytes and Chinese hamster ovary cells. Under these conditions, oxidolanosterol also increases the rate of degradation of reductase protein in these cells. When demethylation is not inhibited, oxidolanosterol treatment produces transcriptional regulation of sterol-sensitive genes in Chinese hamster ovary cells. In contrast to previous findings with the oxygenated C27-sterol, 25-hydroxycholesterol, oxidolanosterol can act as a post-transcriptional regulator in cells starved for mevalonate. These findings are consistent with the hypothesis that oxidolanosterol down-regulates sterol synthesis in a fashion mechanistically distinct from that of C27-sterols.

Animals↗
PubMed1992

Feedback inhibition of polyisoprenyl pyrophosphate synthesis from mevalonate in vitro. Implications for protein prenylation.

The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo.

Animals↗
PubMed1992

Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.

The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p21ras, which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteine-aliphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatin-treated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.

Animals↗