Substrate specificity determinants in the farnesyltransferase β-subunit

Protein prenyltransferases catalyze the covalent attachment of isoprenoid lipids (farnesyl or geranylgeranyl) to a cysteine near the C terminus of their substrates. This study explored the specificity determinants for interactions between the farnesyltransferase of Saccharomyces cerevisiae and its protein substrates. A series of substitutions at amino acid 149 of the farnesyltransferase β-subunit were tested in combination with a series of substitutions at the C-terminal amino acid of CaaX protein substrates Ras2p and a-factor. Efficient prenylation was observed when oppositely charged amino acids were present at amino acid 149 of the yeast farnesyltransferase β-subunit and the C-terminal amino acid of the CaaX protein substrate, but not when like charges were present at these positions. This evidence for electrostatic interaction between amino acid 149 and the C-terminal amino acid of CaaX protein substrates leads to the prediction that the C-terminal amino acid of the protein substrate binds near amino acid 149 of the yeast farnesyltransferase β-subunit.

Interaction of human apurinic endonuclease and DNA polymerase β in the base excision repair pathway

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase β (Polβ) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein–protein contact between Polβ and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polβ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polβ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5′-terminal deoxyribose 5-phosphate by Polβ. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.

Chemical camouflage of antigenic determinants: Stealth erythrocytes

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution–phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal in vivo survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouflage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic β cells) medicine.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The α-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel α-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase

3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (ɛA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only ɛA DNA glycosylase in liver, testes, and kidney; another ɛA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag −/− mice are alkylation sensitive, indicating that Aag −/− mice may be similarly sensitive.

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.

In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen

T cells recognizing poorly displayed self determinants escape tolerance mechanisms and persist in the adult repertoire. The process by which these T cells are primed is not clear, but once activated, they can cause autoimmunity. Here, we show that dendritic cells treated with interleukin 6 (IL-6) process and present determinants from a model native antigen in a qualitatively altered hierarchy, activating T cells in vitro and in vivo against determinants that were previously cryptic because of poor display. IL-6 does not induce conventional maturation of dendritic cells but alters the pH of peripheral, early endosomal compartments and renders the cells more susceptible to killing by chloroquine. Acidification of endosomes by ouabain mimics the effect of IL-6 and allows processing of the same cryptic determinant. These results suggest that cytokines such as IL-6 could initiate and help to propagate an autoimmune disease process by differentiating dendritic cells into a state distinct from that induced by normal maturation.

The Akt kinase: Molecular determinants of oncogenicity

The serine-threonine kinase Akt is a downstream target of phosphoinositide 3-kinase (PI 3-kinase); it is activated by the phosphoinositide 3-phosphate-dependent kinases PDK1 and PDK2. Certain mutated forms of Akt induce oncogenic transformation in chicken embryo fibroblast cultures and hemangiosarcomas in young chickens. This ability to transform cells depends on localization of Akt at the plasma membrane and on the kinase activity of Akt. A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase. Akt is therefore an essential mediator of p3k-induced oncogenicity.

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an α-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R2 = 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

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.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

The common nodABC genes of Rhizobium meliloti are host-range determinants

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA

Nondistorting C4′ backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4′ adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson–Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA–RPA complex may promote damage recognition by monitoring Watson–Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.