Mitochondrial adenine nucleotide translocase is modified oxidatively during aging

The purpose of this study was to test the hypothesis that elevation in protein oxidative damage during the aging process is a targeted rather than a stochastic phenomenon. Oxidative damage to proteins in mitochondrial membranes in the flight muscles of the housefly, manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl antibodies. Adenine nucleotide translocase (ANT) was found to be the only protein in the mitochondrial membranes exhibiting a detectable age-associated increase in carbonyls. The age-related elevation in ANT carbonyl content was correlated with a corresponding loss in its functional activity. Senescent flies that had lost the ability to fly exhibited a relatively higher degree of ANT oxidation and a greater loss of functional activity than their cohorts of the same age that were still able to fly. Exposure of flies to 100% oxygen resulted in an increase in the level of ANT carbonyl content and a loss in its activity. In vitro treatment of mitochondria with a system that generated hydroxyl free radicals caused an increase in ANT carbonyl level and a decrease in ANT exchange activity. ANT was also the only mitochondrial membrane protein exhibiting adducts of the lipid peroxidation product 4-hydroxynonenal. Results of this study indicate that proteins in mitochondrial membranes are modified selectively during aging.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205–2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Production of cyclic peptides and proteins in vivo

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1− and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT− cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT− products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1− cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1− cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1− cells are repaired by illegitimate recombination.

Physical interaction between components of DNA mismatch repair and nucleotide excision repair

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as “bait,” and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair

After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li–Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G1 arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li–Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.

Genetic epidemiology of single-nucleotide polymorphisms

On the causal hypothesis, most genetic determinants of disease are single-nucleotide polymorphisms (SNPs) that are likely to be selected as markers for positional cloning. On the proximity hypothesis, most disease determinants will not be included among markers but may be detected through linkage disequilibrium with other SNPs. In that event, allelic association among SNPs is an essential factor in positional cloning. Recent simulation based on monotonic population expansion suggests that useful association does not usually extend beyond 3 kb. This is contradicted by significant disequilibrium at much greater distances, with corresponding reduction in the number of SNPs required for a cost-effective genome scan. A plausible explanation is that cyclical expansions follow population bottlenecks that establish new disequilibria. Data on more than 1,000 locus pairs indicate that most disequilibria trace to the Neolithic, with no apparent difference between haplotypes that are random or selected through a major disease gene. Short duration may be characteristic of alleles contributing to disease susceptibility and haplotypes characteristic of particular ethnic groups. Alleles that are highly polymorphic in all ethnic groups may be older, neutral, or advantageous, in weak disequilibrium with nearby markers, and therefore less useful for positional cloning of disease genes. Significant disequilibrium at large distance makes the number of suitably chosen SNPs required for genome screening as small as 30,000, or 1 per 100 kb, with greater density (including less common SNPs) reserved for candidate regions.

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3′ end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3′-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki ≈ 5 μM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3′ nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Carbamoyl-phosphate synthetases (CPSs) utilize two molecules of ATP at two internally duplicated domains, B and C. Domains B and C have recently been shown to be structurally [Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M. & Rayment, I. (1997) Biochemistry 36, 6305–6316] and functionally [Guy, H. I. & Evans, D. R. (1996) J. Biol. Chem. 271, 13762–13769] equivalent. We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A/B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoyl-phosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.