Engineering secretable forms of chaperones for immune modulation and vaccine development.

Heat shock proteins are present in almost all intracellular compartments and serve by folding newly synthesized proteins, disassembling unstable proteins, and assisting in the transportation of proteins within the cell. Under certain circumstances they are also present on the cell surface, and can be shed or secreted into the extracellular environment. Although they possess many functional roles, their ability to stimulate innate and antigen-specific immunity have made them attractive candidates for vaccine development. Here, we review some of the approaches that have been used to genetically engineer molecular chaperones for their secretion from tumor cells or targeting them to the plasma membrane of such cells in order to promote anti-tumor responses. Treatment of tumor cells engineered to secrete or display chaperones may be of benefit, particularly in the area of cell-based vaccine development.

Activation of MHC class I, II, and CD40 gene expression by histone deacetylase inhibitors.

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-gamma. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme.

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Interaction of the amino-terminus of an influenza virus protein with mitochondria.

Reye's Syndrome is characterized in part by liver mitochondrial damage. Previous studies in an animal model have demonstrated the presence of viral antigens in hepatocytes, but replication of the virus does not occur. Evidence is presented in this paper for the interaction of an influenza virus protein with mitochondria as a mechanism for mitochondrial damage in Reye's Syndrome. The amino-terminal 22 amino acids of the PB2 protein of influenza A interact with mitochondria alone (5 micrograms/mg mitochondrial protein) and, when conjugated to bovine serum albumin, mediate the binding of BSA to mitochondria (14 micrograms/mg mitochondrial protein).

Identification of putative internalization signals in prion proteins.

Prion proteins were found to contain regions in their cytoplasmic domains that have significant structural homologies to molecular trafficking signals for internalization of membrane proteins. These regions may facilitate the endocytosis of prion proteins, which appears to be a first step in their conversion to the abnormal, amyloidogenic form.

The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.

Isolation and characterization of a protein C activator from tropical moccasin venom.

A protease from the venom of the tropical moccasin (Agkistrodon bilineatus) that activates protein C was purified to homogeneity by ion-exchange and gel permeation chromatography. The purified protease is a glycoprotein, and exhibited a molecular weight of 35,000 and 38,000 in SDS-PAGE under non-reducing and reducing conditions, respectively. The purified protease readily activated human protein C and steady-state kinetic parameters indicated an apparent Km for human protein C of 1.7 microM and an apparent kcat of 0.02 sec-1. Calcium inhibited the activation of human protein C by the venom protease (Ki = 93 microM). Amino-terminal sequence analysis revealed that the tropical moccasin protein C activator was highly homologous to the protein C activator isolated from Southern copperhead venom.

Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties.

Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein of Mr 27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and pI 5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 micrograms/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1 was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 degrees C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7 and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being on Vmax rather than Km. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.

An N-terminal domain of the tetracycline resistance protein increases susceptibility to aminoglycosides and complements potassium uptake defects in Escherichia coli.

Expression of extrachromosomal tet genes increased the susceptibility of gram-negative bacteria to specific aminoglycoside antibiotics. The magnitude of the increase in susceptibility was dependent on the amount and the class of the tet gene product (designated Tet) and the bacterial species in which the tet gene was expressed. Truncated Tet proteins that contained more than the first 33, but not more than the first 97, N-terminal amino acids of Tet also increased the susceptibility to aminoglycosides and complemented the potassium uptake defects in Escherichia coli. The primary structure of this N-terminal Tet fragment has the hydropathic characteristics of a multimeric, transmembrane structure and is highly conserved in three different classes of Tet proteins.

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

Genetic control of the immune response to haemoglobin. IV. Ly-1+ T-cells and appropriate non-H-2 genes are required for in vitro responses to alpha- and beta-subunits of human adult haemoglobin.

Experiments were conducted to determine if non-H2 gene effects could be demonstrated in mice which had been primed to either the alpha-subunit or beta-subunit of human haemoglobin. It was found that C3H.SW (H-2b) and Balb/c (H-2d) mice are low responder mice to alpha-chain of a haemoglobin when compared to H-2 identical B10 (H-2b) and B10.D2(H-2d) mice. B120.S and A.SW (both H-2s) are responsive to beta-chain challenge while Balb/c mice are low responders in contrast to high responder B10.D2 mice. Ly-1+ cells were demonstrated to be required (by cell depletion experiments) for an in vitro T-cell proliferative response to either subunit. In these experiments, Ly-2+ cells were not of crucial importance.

Genetic control of the immune response to haemoglobin. III. Variant A beta (bm12) but not Ae (D2.GD) Ia polypeptides alter immune responsiveness towards the alpha -subunit of human haemoglobin.

Mice bearing the I-A subregion mutation bm12 were immunized and challenged with the alpha -subunit of human adult haemoglobin. Under conditions in which parental B6/Kh mice respond, B6.C-H-2bm12 mice are inhibited nearly 100% in their ability to respond to challenge to the alpha-chain of haemoglobin. D2.GD mice which express a variant Ae (E beta) polypeptide of the I-E subregion can respond as well as B10.D2 mice to both subunits (alpha- and beta-) of haemoglobin. These observations as well as other genetic mapping data confirm the I-A mapping of alpha -chain-specific Ir genes and extend the genetic fine mapping to the A beta gene within the I-A subregion or a combinatorial Ia determinant generated by an interaction of A alpha and A beta. In addition they implicate the Ia.8 specificity in determining immune responsiveness to alpha -chain determinants.

Genetic control of the immune response to haemoglobin. II. Studies using purified alpha-chain and beta-chain as immunogens.

Mice of independent haplotypes and several recombinant inbred strains were immunized with highly purified preparations of either the alpha-chain or beta-chain subunit of human adult haemoglobin. Cells from the sensitized lymph nodes were challenged in vitro with the appropriate subunit (or in some cases both chains) and cell proliferation assessed by 3H-thymidine incorporation. Mice of the H-2b and H-2d haplotypes were high responders to alpha-chain while mice of the H-2f, H-2j, H-2k, H-2r, H-2s, H-2u, and H-2v haplotype were low responders. the low responsivenesss of B10.A(4R) and B 10.MBR and high responsiveness of B10 indicated that the Ir gene(s) determining responsiveness to the alpha-chain subunit resides in the I-A subregion of the mouse major histocompatibility complex. Mice of the H-2d, H-2f, and H-2s haplotypes were high responders and H-2b, H-2j, H-2a, and H-2u haplotype mice were low responders to beta-chain. H-2k, H-2p, H-2r, and H-2v haplotype mice were intermediate responders to beta-chain. The low responsiveness of B10.S(8R) and B10.TL and the high responsivenes of B10.S(9R) mapped the Ir gene(s) to beta-chain to the I-A subregion. Data collected from challenging high responder cells with both subunit indicated that alpha-chain and beta-chain do not crossreact. These results are discussed in reference to earlier observations suggesting that the low responsiveness of some strains of mice to priming and challenging using the intact haemoglobin molecule might be due to a negative regulatory influence mediated by one of the subunits. In the absence of this influence these mice would respond normally.

High yield coupling of peptides to protein carriers.

Coupling of peptides to protein carriers has been achieved at a high level of peptide incorporation. Bovine serum albumin was used as the model carrier protein and several peptides were tested in the coupling reaction. The peptides were coupled to succinylated bovine serum albumin after conversion of its carboxyl side chains to the reactive p-nitrophenyl ester group by reaction with p-nitrophenol/N,N'-dicyclohexylcarbodiimide in anhydrous dimethylformamide. The reaction afforded succinyl-albumin-peptide conjugates that had high level of peptide incorporation (18-35 mol peptide/mol albumin). In addition to the high level of peptide coupling, the reaction avoids the production of polymeric forms of peptide, protein or conjugate.

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

Genetic control of the immune response to haemoglobin. I. Demonstration of separate genetic control of the responses to the alpha- and beta-subunits by in vitro lymphocyte proliferation.

These studies were undertaken to analyse the genetic control of the immune response to an oligomeric protein and the role of individual subunits in the regulation of the response. Human adult haemoglobin (Hb) was selected as a model for these studies because it is a well-characterized protein and its antigenic structure is being determined in our laboratories. Mice of various congenic strains were immunized with Hb and the lymph node cells from Hb-primed mice were challenged in vitro with Hb, and its alpha-chain and beta-chains as well as an appropriate control antigen. Lymphocyte proliferation was determined by 3H-thymidine incorporation. The data collected indicated that mice of the H-2b and H-2d haplotypes were high responders while H-2k, H-2s, H-2q and H-2j haplotype mice were low responders to Hb. Studies with H-2 recombinant strains indicated that the immune response to Hb and its subunits is determined by genes in the I-A subregion and the D end of the H-2 complex. The significance of these findings in terms of control and regulation of the overall response to native Hb are discussed.

Nearest-neighbour analysis of myoglobin antigenic sites. Nearest-neighbour residues whose replacement can alter the environment of binding-site residue(s) and thus change their characteristics and binding capability.

By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).