Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis.

The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E. coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

cDNA cloning and expression of pokeweed antiviral protein from seeds in Escherichia coli and its inhibition of protein synthesis in vitro.

Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). We isolated, sequenced and characterized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds. The cDNA of PAP-S consists of 1249 nucleotides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%). It is known from literature that PAP-S is more active in inhibiting protein synthesis than other members of the PAP family. Therefore, the cDNA of PAP-S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified from spring leaves. In a rabbit translation system, the median inhibitory concentrations (IC50) of recombinant PAP-S and native PAP were determined as 0.07 and 0.29 nM, respectively. Although the PAP-S protein in seeds is glycosylated, PAP-S can be expressed in Escherichia coli in a very active form, indicating that post-translational modification in pokeweed does not seem to alter its ability to inhibit protein synthesis.

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli.

Three distinct ribosome-inactivating proteins (RIPs) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

The cysteine residues in the cytoplasmic tail of CD8 alpha are required for its coreceptor function.

The cytoplasmic segment of the CD8 alpha polypeptide includes both a cysteine-containing motif that is required for its association with the tyrosine kinase p56lck, and two serine residues which are likely to be phosphorylated and involved in inside-out signaling phenomena. To determine the relative importance of these residues for CD8 function, a mouse T cell hybridoma expressing a T cell receptor specific for the class I major histocompatibility product H-2Kb was transfected with a set of CD8 alpha chain genes encoding polypeptides in which the cytoplasmic cysteine or serine residues were substituted with alanine. When challenged with Kb-transfected L cells, T cell transfectants expressing CD8 alpha beta or CD8 alpha alpha dimers with substituted cytoplasmic serine residues responded nearly as well as wild-type CD8 transfectants. In marked contrast, the CD8 alpha polypeptides bearing substitutions of both cytoplasmic cysteine residues were totally impaired in their ability to complement the co-expressed T cell receptor.

Role of CD3 delta in surface expression of the TCR/CD3 complex and in activation for killing analyzed with a CD3 delta-negative cytotoxic T lymphocyte variant.

The TCR is composed of two chains (alpha/beta) containing variable regions associated at the cell surface with invariant chains (CD3 gamma-, delta-, epsilon-, and zeta/eta chains). The latter control assembly and surface expression of the TCR/CD3 complex, as well as its cytoplasmic association with signal transduction relays. In differentiated CTL, stimulation through the TCR leads to the transcriptional activation of genes coding secreted cytokines such as gamma-IFN as well as transcription-independent activation of the lytic machinery. It is not known which of the CD3 components is necessary to transduce the required signals. CD3 gamma- and delta-chains have high sequence homology, in particular in their cytoplasmic domain, and it has been proposed that alpha beta gamma epsilon zeta and alpha beta delta, epsilon zeta may be expressed and function in signal transduction independently. Here, we characterize a CTL clone that has selectively lost expression of the CD3 delta mRNA. This results in expression of partial CD3 complexes devoid of TCR alpha beta chains at the surface of the clone, which are not functional for activation of cytolysis or for gamma-IFN production. Transfection of the clone with either the native or a cytoplasmic exon-deleted CD3 delta gene restores full TCR/CD3 surface expression as well as Ag- or CD3-mediated activation for killing and for gamma-IFN production, indicating that the CD3 delta chain is essential for surface expression of the TCR alpha beta, but that the CD3 delta cytoplasmic portion is not required either for complex assembly or for signal transduction involved in the functions studied.

The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules.

Recent studies have demonstrated that the CD3-zeta subunit of the T cell antigen receptor (TCR) complex is involved in signal transduction. However, the function of the remaining invariant subunits, CD3-gamma, -delta, and epsilon, is still poorly understood. To examine their role in TCR function, we have constructed TCR/CD3 complexes devoid of functional zeta subunit and showed that they are still able to trigger the production of interleukin-2 in response to antigen or superantigen. These data, together with previous results, indicate that the TCR/CD3 complex is composed of at least two parallel transducing units, made of the gamma delta epsilon and zeta chains, respectively. Furthermore, the analysis of partially truncated zeta chains has led us to individualize a functional domain that may have constituted the building block of most of the transducing subunits associated with antigen receptors and some Fc receptors.

Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding.

In studies on the promoter-inhibitory effect of sequence-specific DNA methylations, the late E2A promoter of human adenovirus type 2 (Ad2) has been used as an experimental tool. Upon the in vitro methylation of 5'-CCGG-3' (HpaII) sequences at nucleotides +24, +6 and -215 relative to nucleotide + 1, the site of transcriptional initiation, promoter inhibition or inactivation has been demonstrated in transient expression tests in Xenopus laevis oocytes (Langner et al., 1984), in mammalian cells (Langner et al., 1986), after the genomic fixation of the promoter in conjunction with a reporter gene in mammalian cells (Müller & Doerfler, 1987), and in a cell-free transcription system employing nuclear extracts of human HeLa cells (Dobrzanski et al., 1988). Possible explanations for the inhibitory effect of three 5-methyldeoxycytidine (5-mC) residues in a promoter sequence are structural alterations in DNA or the positive or negative modulation of the sequence-specific binding of proteins. This modulation could be indirect at the level of protein-protein interactions. A synthetic oligodeoxyribonucleotide of 50 base-pairs (bp) or a restriction endonuclease fragment of 73 bp in length, which comprised the +24 and +6 5'-CCGG-3' sequences of the late E2A promoter, were methylated or hemimethylated at these two sites, or were left unmethylated and were subsequently incubated with a partly purified (heparin-Sepharose) nuclear extract of human HeLa cells. Protein binding was monitored by electrophoretic migration delay of the 32P-labeled 50 bp oligodeoxyribonucleotide or the 73 bp fragment on polyacrylamide gels. The formation of one of the DNA-protein complexes in this analysis was compromised when 5'-CCGG-3' methylated oligodeoxyribonucleotides were used in the binding assays. Similar results were obtained when the 50 bp oligodeoxyribonucleotide was hemimethylated in either complement.The formation of the same complex could also be obliterated by adding the same non-methylated oligodeoxyribonucleotide as competitor to the reaction mixture. The methylated oligodeoxyribonucleotide did not act as a competitor, nor did a randomly composed oligodeoxyribonucleotide of identical length. The results show that protein binding is abolished by methylation of those sequences in the late E2A promoter whose methylation inhibits promoter function. The abrogation of protein binding has been observed with a 50 bp or 73 bp fragment. With a 99 bp or a 377 bp fragment, binding differences between the unmethylated and the 5'-CCGG-3' methylated late E2A promoter are not apparent.

Promoter inactivation or inhibition by sequence-specific methylation and mechanisms of reactivation.

In studies on adenovirus promoters, predominantly on the late E2A promoter of adenovirus type 2 (Ad2), we have demonstrated by a number of experimental approaches that the sequence-specific methylation of three 5'-CCGG-3' sequences inactivates this promoter. Recently, we have developed a cell-free transcription system in which the methylation-inactivation of eukaryotic promoters can be studied in detail. It has also been shown that methylation-caused promoter inactivation can be reversed by the 289 amino acid E1A protein of Ad2 or of adenovirus type 5. In the presence of this protein with a transactivating effect, transcription is initiated at the authentic cap site of the methylated late E2A promoter. A similar reactivation of the methylated late E2A promoter can also be effected by a cis-acting genetic element, i.e., the strong enhancer of human cytomegalovirus. Further studies will be directed toward the biochemical mechanisms of promoter silencing by sequence-specific methylations.

Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system.

Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific promoter methylations necessitated the development of a cell-free transcription system that responded to in vitro promoter methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, the activities of two adenovirus type 2 promoters in the nonmethylated and methylated forms were compared. The late E2A promoter in vitro methylated at three 5'-CCGG-3' (HpaII) sequences at nucleotides -215, +6, and +24, or the major late promoter in vitro methylated at nucleotide -52 in the 5'-CCGG-3' sequence or at nucleotide -13 in the 5'-GCGC-3' (HhaI) sequence exhibited strikingly lower activities than did the nonmethylated constructs or exhibited no activity at all. The designated nucleotide positions were calculated relative to the cap sites of the two promoters. Upon in vitro transcription, the methylation pattern of the E2A late promoter was shown to be stable. For the inhibitory effects by sequence-specific methylations to be elicited, circular templates had to be used, the DNA titers had to be at critical levels for each extract, and high protein concentrations had to be maintained. When a template mixture of the nonmethylated major late promoter and the 5'-CCGG-3' methylated late E2A promoter of adenovirus type 2 DNA was used, the major late promoter was active and the methylated late E2A promoter was inhibited or inactivated. Activity levels of the two different promoters could be assessed simultaneously in the same assay due to differences in lengths between the products of transcription from the late E2A and major late promoters. Thus inhibition in the cell-free system could be proven to be specific for the methylated promoter. We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and somehow participate in their inactivation.

Specific factors binding to the late E2A promoter region of adenovirus type 2 DNA: no apparent effects of 5'-CCGG-3' methylation.

In the regulation of eukaryotic gene expression, the interactions of several protein factors with specific signals in the promoter sequence can play a decisive role. In addition, the methylation of specific promoter sequences causes the long-term inactivation of eukaryotic promoters. The function of the regulatory signal 5-methylcytidine is complex and can be overcome by a number of factors, e.g., by the E1 proteins of adenoviruses. We studied the inactivation of the late E2A promoter of adenovirus type 2 (Ad2) DNA by the methylation of three 5'-CCGG-3' sequences at positions -215, +5, and +23, relative to one of the cap sites of this promoter. One would like to understand the mechanisms by which 5-methylcytidine residues are capable of interfering with regulatory functions in the late E2A promoter of Ad2. We have identified six different promoter sequences by DNase I protection analyses, and have shown that these sites bind specifically to host proteins (binding sites I-VI). Binding of these factors to unmethylated or 5'-CCGG-3' methylated late E2A promoter sequences was compared by gel migration delay assay or DNase I protection (footprinting) analyses. Protein binding does not appear to be affected by late E2A promoter methylation. Even after partial purification of some of these factors by chromatography on heparin-Sepharose, differences in binding to the unmethylated and the 5'-CCGG-3' methylated promoter were not observed. Even though striking differences in host factor binding were not detectable at late E2A promoter sites, it is conceivable that the functionality of promoter-bound host proteins is altered when the three 5'-CCGG-3' sequences are methylated.