Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.

Nitrogen-regulated Ubiquitination of the Gap1 Permease of Saccharomyces cerevisiae

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4+ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4+-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4+ to mutants defective in endocytosis.

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin, an analog 500-1000 times more potent: structure-activity relationship of daunosamine-modified derivatives of doxorubicin.

A convenient, high yield conversion of doxorubicin to 3'-deamino-3'-(2''-pyrroline-1''-yl)doxorubicin is described. This daunosamine-modified analog of doxorubicin is 500-1000 times more active in vitro than doxorubicin. The conversion is effected by using a 30-fold excess of 4-iodobutyraldehyde in anhydrous dimethylformamide. The yield is higher than 85%. A homolog of this compound, 3'-deamino-3'-(1'',3''-tetrahydropyridine-1''-yl)doxorubicin, was also synthesized by using 5-iodovaleraldehyde. In this homolog, the daunosamine nitrogen is incorporated into a six- instead of a five-membered ring. This analog was 30-50 times less active than its counterpart with a five-membered ring. A similar structure-activity relationship was found when 3'-deamino-3'-(3''-pyrrolidone-1''-yl)doxorubicin (containing a five-membered ring) and 3'-deamino-3'-(3''-piperidone-1''-yl)doxorubicin (with a six-membered ring) were tested in vitro, the former being 5 times more potent than the latter. To further elucidate structure-activity relationships, 3'-deamino-3'-(pyrrolidine-1''-yl)doxorubicin, 3'-deamino-3'-(isoindoline-2''-yl)doxorubicin, 3'-deamino-3'-(2''-methyl-2''-pyrroline-1''-yl)doxorubicin, and 3'-deamino-3'-(3''-pyrroline-1''-yl)doxorubicin were also synthesized and tested. All the analogs were prepared by using reactive halogen compounds for incorporating the daunosamine nitrogen of doxorubicin into a five- or six-membered ring. These highly active antineoplastic agents can be used for incorporation into targeted cytotoxic analogs of luteinizing hormone-releasing hormone intended for cancer therapy.

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1

The ubiquitin-like protein RUB1 is conjugated to target proteins by a mechanism similar to that of ubiquitin conjugation. Genetic studies in Arabidopsis thaliana have implicated the RUB-conjugation pathway in auxin response. The first step in the pathway is RUB activation by a bipartite enzyme composed of the AXR1 and ECR1 proteins. Ubiquitin activation is an ATP-dependent process that involves the formation of an AMP-ubiquitin intermediate. Here we show that RUB activation by AXR1-ECR1 also involves formation of an AMP-RUB intermediate and that this reaction is catalyzed by the ECR1 subunit alone. In addition, we identified an Arabidopsis protein called RCE1 that is a likely RUB-conjugating enzyme. RCE1 works together with AXR1-ECR1 to promote formation of a stable RUB conjugate with the Arabidopsis cullin AtCUL1 in vitro. Using a tagged version of RUB1, we show that this modification occurs in vivo. Because AtCUL1 is a component of the ubiquitin protein ligase SCFTIR1, a complex that also functions in auxin response, we propose that RUB modification of AtCUL1 is important for auxin response.

Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C2-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

Yeast Cycloheximide-resistant crl Mutants Are Proteasome Mutants Defective in Protein Degradation

In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3–2, had been found to be suppressed by a mutation, SCL1–1, which resided in an α-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3–2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1–1 suppressor mutation causes partial restoration of crl3–2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1–1, pre2–1, pre3–1, pre4–1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.

Interaction of the poliovirus receptor with poliovirus

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-Å resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus “canyon” and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.

Inhibition of β-catenin-mediated transactivation by cadherin derivatives

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of β-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with β-catenin and protected it from degradation. N-cadherin directed β-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of β-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with β-catenin in the nucleus. Cotransfection of β-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in SW480 cells relocated β-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N- or E-cadherin colocalized with β-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking β-catenin–LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized β-catenin and reduced its transactivation potential. These results indicate that β-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit β-catenin degradation and efficiently block its transactivation capacity.