HIV-1 GP120-mediated immune suppression and lymphocyte destruction in the absence of viral infection

The magnitude of immunologic defects observed in HIV-1-infected individuals before the development of overt AIDS is disproportionately high in comparison to the levels of infectious virus in these patients--suggesting that factors other than direct virus-induced cytopathology may be involved. With this in mind, we investigated the immunologic consequences of the interaction between purified HIV-1 gp120 and the CD4 molecules expressed by uncommitted as well as Ag-specific lymphocytes. HIV-1 gp120 exhibited a dose-dependent immunosuppressive effect on: 1) Ag-driven proliferation of cloned CD4+ lymphocytes, 2) OKT3-driven proliferation of cloned CD4+ lymphocytes, and 3) cytolytic activity of CD4+, EBV-specific CTL. Thus, HIV-1 gp120 can, in a manner similar to OKT4A antibodies, suppress T cell activation and the expression of cytolytic activities through its interaction with CD4. Additionally, activated CD4+ lymphoblasts can be rendered susceptible to immune cytolysis by virtue of their binding of purified gp120. This "targeting" of activated lymphoblasts can occur with levels of gp120 far below that which is needed to saturate all OKT4A-defined CD4 epitopes. Adsorbed gp120 could be demonstrated on the surface of these cells for up to 12 h, a sufficient time for interaction with host cytolytic elements. The data from these in vitro modeling experiments highlight one of many potential mechanisms of HIV-1 induced immunosuppression and lymphocyte destruction that can occur in the absence of infectious virus and that is based on the unique interaction between HIV-1 gp120 and its cellular receptor, CD4.     

Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures

Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 ± 5000, 105,000 ± 5000, and 94,000 ± 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M_r, as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K_m values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and α-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase

Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS.     

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41.

Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537-10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence.     

Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1'' binding site.

Structures of the blood clotting enzyme thrombin complexed with hirugen and two active site inhibitors, RWJ-50353 10080(N-methyl-D-phenylalanyl-N-[5-[(aminoiminomethyl)amino]-1- [[(2-benzothiazolyl)carbonyl]butyl]-L-prolinamide trifluoroacetate hydrate) and RWJ-50215 (N-[4-(aminoiminomethyl)amino-1-[2- (thiazol-2-ylcarbonylethyl)piperidin- 1-ylcarbonyl]butyl]-5-(dimethylamino)naphthalenesulfonamide trifluoroacetate hydrate), were determined by x-ray crystallography. The refinements converged at R values of 0.158 in the 7.0-2.3-A range for RWJ-50353 and 0.155 in the 7.0-1.8-A range for RWJ-50215. Interactions between the protein and the thiazole rings of the two inhibitors provide new valuable information about the S1' binding site of thrombin. The RWJ-50353 inhibitor consists of an S1'-binding benzothiazole group linked to the D-Phe-Pro-Arg chloromethyl ketone motif. Interactions with the S1-S3 sites are similar to the D-phenylalanyl-prolyl-arginyl chloromethylketone structure. In RWJ-50215, a S1'-binding 2-ketothiazole group was added to the thrombin inhibitor-like framework of dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The geometry at the S1-S3 sites here is also similar to that of the parent compound. The benzothiazole and 2-ketothiazole groups bind in a cavity surrounded by His57, Tyr60A, Trp60D, and Lys60F. This location of the S1' binding site is consistent with previous structures of thrombin complexes with hirulog-3, CVS-995, and hirutonin-2 and -6. The ring nitrogen of the RWJ-50353 benzothiazole forms a hydrogen bond with His57, and Lys60F reorients because of close contacts. The oxygen and nitrogen of the ketothiazole of RWJ-50215 hydrogen bond with the NZ atom of Lys60F.