Molecular characterization of CD40 signaling intermediates.

Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.

High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization.

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.

Crystallographic analysis of CD40 recognition and signaling by human TRAF2.

Tumor necrosis factor receptor superfamily members convey signals that promote diverse cellular responses. Receptor trimerization by extracellular ligands initiates signaling by recruiting members of the tumor necrosis factor receptor-associated factor (TRAF) family of adapter proteins to the receptor cytoplasmic domains. We report the 2.4-A crystal structure of a 22-kDa, receptor-binding fragment of TRAF2 complexed with a functionally defined peptide from the cytoplasmic domain of the CD40 receptor. TRAF2 forms a mushroom-shaped trimer consisting of a coiled coil and a unique beta-sandwich domain. Both domains mediate trimerization. The CD40 peptide binds in an extended conformation with every side chain in contact with a complementary groove on the rim of each TRAF monomer. The spacing between the CD40 binding sites on TRAF2 supports an elegant signaling mechanism in which trimeric, extracellular ligands preorganize the receptors to simultaneously recognize three sites on the TRAF trimer.

CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.

Tumor necrosis factor receptor-associated factors (TRAFs) associate with the CD40 cytoplasmic domain and initiate signaling after CD40 receptor multimerization by its ligand. We used saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3 and TRAF6 binding sequences in CD40 to finely map residues involved in CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site demonstrated more amino acid sequence flexibility and could be optimized. Point mutations that eliminated or enhanced binding of TRAFs to one or both sites were made in CD40 and tested in quantitative CD40-TRAF binding assays. Sequences flanking the core TRAF binding sites were found to modulate TRAF binding, and the two TRAF binding sites were not independent. Cloned stable transfectants of human embryonic kidney 293 cells that expressed wild type CD40 or individual CD40 mutations were used to demonstrate that both TRAF binding sites were required for optimal NF-kappaB and c-Jun N-terminal kinase activation. In contrast, p38 mitogen-activated protein kinase activation was primarily dependent upon TRAF6 binding. These studies suggest a role in CD40 signaling for competitive TRAF binding and imply that CD40 responses reflect an integration of signals from individual TRAFs.

CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization.

CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.

Characterization of the human cytomegalovirus UL105 gene and identification of the putative helicase protein.

We have characterized the transcription unit for the human cytomegalovirus UL105 gene and identified its putative protein product. The UL105 gene product is proposed to mediate helicase activity in the assembled helicase-primase complex. The two other putative proteins in this complex are the gene products of UL102 (primase-associated factor) and UL70 (primase). Using Northern blot analysis we have determined that the UL105 transcript is a 3.4-kb message that can be detected as early as 24 hr postinfection in the presence of phosphonoformic acid but not in the presence of cycloheximide. Subsequent primer-extension analysis showed a transcriptional start site upstream of a consensus TATA sequence and just downstream of a CCAAT box sequence motif. In addition, we have identified an infected cell protein with an approximate molecular weight (M(r)) of 110 kDa using anti-peptide antiserum. This same antiserum detected proteins of the same M(r) as those produced from two expression systems where the protein from the 981-amino-acid UL105 open reading frame was overexpressed, suggesting that the ATG located at nt 151,850 of the genomic sequence is utilized in the context of the virus genome.

Identification of the primase active site of the herpes simplex virus type 1 helicase-primase.

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase composed of the products of the three DNA replication-specific genes UL5, UL8, and UL52 (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). The UL5 and UL52 products constitute a heterodimeric subassembly of the holoenzyme that contains both helicase and primase activities (Calder, J. M., and Stow, N. D. (1990) Nucleic Acids Res. 18, 3573-3578; Dodson, M. S., and Lehman, I. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1105-1109). The role of the UL52 product in the active HSV-1 helicase-primase was examined. A sequence located between residues 610 and 636 on the UL52 protein was found to be conserved among the UL52 homologues of eight herpesviruses. The carboxyl-terminal portion of this conserved sequence consisted of two Asp residues separated by a variable hydrophobic amino acid residue and is analogous to the divalent metal-binding site of DNA polymerases and several DNA primases. This motif has been designated the herpesvirus primase DXD motif. To study the role of the HSV-1 primase DXD motif in primase action, three site-directed changes were introduced into the UL52 gene. The helicase activity of the recombinant holoenzymes was unaffected by any of the introduced changes. Changing either of the two Asp residues that constitute the divalent metal-binding site (Asp628 or Asp630) to Ala dramatically reduced the primase activity of the HSV-1 helicase-primase holoenzyme in vitro, whereas alteration of the nearby conserved residue Asn624 to Gly had minimal effect. Therefore, in the three-subunit HSV-1 helicase-primase, the UL52 product provides at least a part of the primase catalytic site.

Inhibition of herpes simplex virus type 1 helicase-primase by (dichloroanilino)purines and -pyrimidines.

Herpes simplex virus type 1 (HSV1) encodes a heterotrimeric helicase-primase comprised of the products of three of the seven DNA replication-specific genes. Several dihalo-substituted derivatives of N2-phenylguanines and 2-anilinoadenines weakly inhibited the intrinsic DNA-dependent NTPase activity of the HSV1 helicase-primase, and these compounds inhibited the DNA-unwinding activity of the enzyme. The primase activity of the enzyme was strongly inhibited by 3,4- and 3,5-dichloroanilino derivatives of adenine and 2-aminopyrimidines. These compounds and nucleoside analogs of 2-(3,5-dichloroanilino)purines inhibited viral DNA synthesis in HSV1-infected HeLa cells in culture but also inhibited cellular DNA synthesis, likely as a result of inhibition of cellular primase and/or DNA polymerases.

Herpes simplex-1 helicase-primase. Identification of two nucleoside triphosphatase sites that promote DNA helicase action.

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase comprised of the products of the UL5, UL8, and UL52 genes (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). A steady state kinetic analysis of the enzyme isolated from HSV-1-infected CV-1 cells or insect cells expressing the enzyme after infection with recombinant baculoviruses has shown it to possess two sites capable of hydrolyzing nucleoside triphosphates in a DNA-dependent manner. One site (Site I) hydrolyzes both ATP and GTP; the second (Site II) hydrolyzes only ATP. These two sites are contained within a subassembly of the helicase-primase formed by coexpression of the UL5 and UL52 genes in insect cells. Sites I and II are activated by separate DNA effector sites, both of which support DNA helicase action. These findings are likely to be of importance in understanding how helicases in general catalyze the unwinding of duplex DNA and, in particular, how the helicase-primase functions at the HSV-1 replication fork.

Herpes simplex virus-1 helicase-primase. Physical and catalytic properties.

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of the products of the UL5, UL8, and UL52 genes (Crute, J. J., Tsurumi, T., Zhu, L., Weller, S. K., Olivo, P. D., Challberg, M. D., Mocarski, E. S. and Lehman, I. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2186-2189). Further characterization of the three-subunit enzyme isolated from HSV-1-infected CV-1 cells shows it to be a heterotrimer, consisting of one polypeptide encoded by each of the UL5, UL8, and UL52 genes. Analysis of the primase and helicase components of the HSV-1 helicase-primase has shown that the primase component synthesizes oligoribonucleotide primers 8-12 nucleotides in length. The helicase component unwinds duplex DNA substrates at the rate of about two nucleotides/s, but only in the presence of the HSV-1-encoded single-stranded DNA binding protein. Thus, the HSV-1 helicase-primase contains the requisite enzymatic activities that permit it to function at the viral replication fork.

The herpes simplex virus 1 origin binding protein: a DNA helicase.

A recombinant herpes simplex 1 origin binding protein, the product of the herpes UL9 gene, has been overexpressed in mammalian cells and purified to near homogeneity. The origin binding protein shows DNA-dependent nucleoside 5'-triphosphatase and DNA helicase activities in addition to its origin binding activity. The ability to hydrolyze nucleoside 5'-triphosphates is influenced strongly by the structure and sequence of the DNA cofactor. The properties of the recombinant origin binding protein are identical to those of the protein synthesized in herpes simplex 1-infected mammalian cells.

Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells.

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of three polypeptides encoded by the UL5, UL8, and UL52 genes (Crute, J.J., Tsurumi, T., Zhu, L., Weller, S.K., Olivo, P.D., Challberg, M.D., Mocarski, E.S., and Lehman, I.R. (1989) Proc. Natl. Acad, Sci, U.S.A. 86, 2186-2189). To obtain sufficient quantities of the enzyme for study, we have overexpressed the three genes using the baculovirus expression system. We find that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene. The recombinant enzyme which we have purified to near homogeneity from the insect cells has a molecular weight of 270,000 and is composed of the three polypeptides encoded by the UL5, UL8, and UL52 genes. The enzyme possesses DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are essentially identical to the enzyme isolated from HSV-1-infected cells.

Herpes simplex-1 DNA polymerase. Identification of an intrinsic 5'----3' exonuclease with ribonuclease H activity.

The herpes simplex virus-1 DNA polymerase is a heterodimer of Mr 190,000 which consists of the products of the UL30 (Pol) and UL42 genes. The 136-kilodalton Pol gene product contains an intrinsic ribonuclease H activity that specifically degrades RNA.DNA heteroduplexes or duplex DNA substrates in the 5'----3' direction. It can therefore catalyze the excision of the RNA primers that initiate the synthesis of Okazaki fragments at a replication fork during herpes DNA replication.

Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.

Properties of two forms of DNA polymerase delta from calf thymus.

Purified calf thymus DNA polymerases delta I and II each have an associated 3' to 5' exonuclease but otherwise resemble DNA polymerase alpha in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, J. J., Wahl, A. F., & Bambara, R. A. (1986) Biochemistry 25, 26-36]. Here we demonstrate a functional association of polymerase and exonuclease with each delta form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase delta I but does not appear to be removable from DNA polymerase delta II. Polymerases delta I, delta II, and alpha are equally sensitive to the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase alpha and delta II, DNA polymerase delta I has intermediate sensitivity to 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) or N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The activity of the DNA primase of the delta II enzyme is insensitive to BuAdATP whereas 1.0 microM of this inhibitor will decrease the activity of the DNA primase of the alpha and delta I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase alpha are only slightly inhibitory to DNA polymerase delta I and are ineffective at inhibiting DNA polymerase delta II. DNA polymerase delta II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA.

The beta subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits.

We have previously demonstrated that the addition of a stoichiometric excess of the beta subunit of Escherichia coli DNA polymerase III holoenzyme to DNA polymerase III or holoenzyme itself can lead to an ATP-independent increase in the processivity of these enzyme forms (Crute, J. J., LaDuca, R. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1983) J. Biol. Chem. 258, 11344-11349). Here, we show that the beta subunit can interact directly with the catalytic core of the holoenzyme, DNA polymerase III, generating a new form of the enzyme with enhanced catalytic and processive capabilities. The addition of saturating levels of the beta subunit to the core DNA polymerase III enzyme results in as much as a 7-fold stimulation of synthetic activity. Two populations of DNA products were generated by the DNA polymerase III X beta enzyme complex. Short products resulting from the addition of 5-10 nucleotides/primer fragment were generated by DNA polymerase III in the presence and absence of added beta subunit. A second population of much longer products was generated only in beta-supplemented DNA polymerase III reactions. The DNA polymerase III-beta reaction was inhibited by single-stranded DNA binding protein and was unaffected by ATP, distinguishing it from the holoenzyme-catalyzed reaction. Complex formation of the DNA polymerase III core enzyme with beta increased the residence time of the enzyme on synthetic DNA templates. Our results demonstrate that the beta stimulation of DNA polymerase III can be attributed to a more efficient and highly processive elongation capability of the DNA polymerase III X beta complex. They also prove that at least part of beta's normal contribution to the DNA polymerase III holoenzyme reaction takes place through interaction with DNA polymerase III core enzyme components to produce the essential complex necessary for efficient elongation in vivo.

Inhibition of mammalian DNA polymerases by hematoporphyrin derivative and photoradiation.

Hematoporphyrin derivative (HPD) plus photoradiation caused the inactivation of DNA polymerases from calf thymus and R3230AC rat mammary tumor. Photosensitization of purified DNA polymerase-alpha as well as two forms of DNA polymerase-delta (I and II) from calf thymus were evaluated. Although all polymerase enzyme forms were inactivated at 70 micrograms HPD/ml, DNA polymerase-delta II was the most sensitive, displaying a 90% inactivation under conditions that did not cause significant inactivation of the other polymerase forms. Unlike DNA polymerase-alpha, the delta-forms have an associated 3'- to 5'-exonuclease activity. The exonuclease associated with DNA polymerase-delta II was uniquely sensitive to a low level of HPD and light exposure. DNA polymerase-delta II can be distinguished from other polymerase forms in cell extracts by its relative insensitivity to the polymerase inhibitor N2-(p-n-butylphenyl)deoxyadenosine 5'-triphosphate. In cytosols prepared from calf thymus and R3230AC rat mammary tumors, DNA polymerase-delta II was preferentially inhibited by HPD plus light. Furthermore, in experiments in which tumor-bearing rats were administered HPD prior to preparation of tumor cytosols, DNA polymerase-delta II was specifically inactivated by exposure to light. These results are discussed in view of their possible role in cancer therapy, and the potential use of HPD as a specific inhibitory agent of DNA polymerase-delta II is suggested.

Purification and characterization of two new high molecular weight forms of DNA polymerase delta.

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.