A unified theory of carcinogenesis based on order–disorder transitions in DNA structure as studied in the human ovary and breast

Fourier transform-infrared/statistics models demonstrate that the malignant transformation of morphologically normal human ovarian and breast tissues involves the creation of a high degree of structural modification (disorder) in DNA, before restoration of order in distant metastases. Order–disorder transitions were revealed by methods including principal components analysis of infrared spectra in which DNA samples were represented by points in two-dimensional space. Differences between the geometric sizes of clusters of points and between their locations revealed the magnitude of the order–disorder transitions. Infrared spectra provided evidence for the types of structural changes involved. Normal ovarian DNAs formed a tight cluster comparable to that of normal human blood leukocytes. The DNAs of ovarian primary carcinomas, including those that had given rise to metastases, had a high degree of disorder, whereas the DNAs of distant metastases from ovarian carcinomas were relatively ordered. However, the spectra of the metastases were more diverse than those of normal ovarian DNAs in regions assigned to base vibrations, implying increased genetic changes. DNAs of normal female breasts were substantially disordered (e.g., compared with the human blood leukocytes) as were those of the primary carcinomas, whether or not they had metastasized. The DNAs of distant breast cancer metastases were relatively ordered. These findings evoke a unified theory of carcinogenesis in which the creation of disorder in the DNA structure is an obligatory process followed by the selection of ordered, mutated DNA forms that ultimately give rise to metastases.

Crystal structure of the BTB domain from PLZF

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

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.

The crystal structure of human interferon β at 2.2-Å resolution

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of α and β IFNs, we have determined the crystal structure of glycosylated human IFN-β at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-β and human IFN-α2b but displays several distinct structural features. Like human IFN-α2b, human IFN-β contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-β dimers. However, unlike the human IFN-α2b dimer, in which homologous surfaces form the interface, human IFN-β dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-β at specific exposed residues is discussed. A potential role of ligand–ligand interactions in the conformational assembly of IFN receptor components is discussed.

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.

Inhibition of gene expression in human cells through small molecule-RNA interactions

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from d-and l-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC50 of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation

The structure of truncated human apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein, has been determined at 4-Å resolution. The crystals comprise residues 44–243 (exon 4) of apo A-I, a fragment that binds to lipid similarly to intact apo A-I and that retains the lipid-bound conformation even in the absence of lipid. The molecule consists almost entirely of a pseudo-continuous, amphipathic α-helix that is punctuated by kinks at regularly spaced proline residues; it adopts a shape similar to a horseshoe of dimensions 125 × 80 × 40 Å. Four molecules in the asymmetric unit associate via their hydrophobic faces to form an antiparallel four-helix bundle with an elliptical ring shape. Based on this structure, we propose a model for the structure of apo A-I bound to high density lipoprotein.

Interaction of Class I Human Leukocyte Antigen (HLA-I) Molecules with Insulin Receptors and Its Effect on the Insulin-Signaling Cascade

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, β2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

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.

NMR solution structure of the human prion protein

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90–230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered “tail.” The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–228 and a short anti-parallel β-sheet comprising the residues 128–131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219–228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219–226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.