Postmortem proteomic analysis in human amygdala of drug addicts: possible impact of tubulin on drug-abusing behavior.

Besides the ventral tegmental area and the nucleus accumbens as the most investigated brain reward structures, several reports about the relation between volume and activity of the amygdala and drug-seeking behavior have emphasized the central role of the amygdala in the etiology of addiction. Considering its proposed important role and the limited number of human protein expression studies with amygdala in drug addiction, we performed a human postmortem proteomic analysis of amygdala tissue obtained from 8 opiate addicts and 7 control individuals. Results were validated by Western blot in an independent postmortem replication sample from 12 opiate addicts compared to 12 controls and 12 suicide victims, as a second "control sample". Applying 2D-electrophoresis and MALDI-TOF-MS analysis, we detected alterations of beta-tubulin expression and decreased levels of the heat-shock protein HSP60 in drug addicts. Western blot analysis in the additional sample demonstrated significantly increased alpha- and beta-tubulin concentrations in the amygdala of drug abusers versus controls (P = 0.021, 0.029) and to suicide victims (P = 0.006, 0.002). Our results suggest that cytoskeletal alterations in the amygdala determined by tubulin seem to be involved in the pathophysiology of drug addiction, probably via a relation to neurotransmission and cellular signaling. Moreover, the loss of neuroprotection against stressors by chaperons as HSP60 might also contribute to structural alteration in the brain of drug addicts. Although further studies have to confirm our results, this might be a possible pathway that may increase our understanding of drug addiction.

The amino acid sequence of ovocleidin 17, a major protein of the avian eggshell calcified layer.

The amino acid sequence of ovocleidin 17, a major protein of the chicken eggshell calcified layer, contains 142 amino acids including 2 phosphorylated serines. Data base searches show that ovocleidin belongs to a heterogeneous group of proteins consisting of a single C-type lectin domain (CTL). The most similar sequences with an average of 30% identical amino acids were those of pancreatic stone protein (lithostathine) and lectins and anticoagulant proteins from snake venom.

Activity-dependent regulation of alternative splicing patterns in the rat brain.

Alternative splicing plays an important role in the expression of genetic information. Among the best understood alternative splicing factors are transformer and transformer-2, which regulate sexual differentiation in Drosophila. Like the Drosophila genes, the recently identified mammalian homologues are subject to alternative splicing. Using an antibody directed against the major human transformer-2 beta isoform, we show that it has a widespread expression in the rat brain. Pilocarpine-induced neuronal activity changes the alternative splicing pattern of the human transformer-2-beta gene in the brain. After neuronal stimulation, a variant bearing high similarity to a male-specific Drosophila tra-2179 isoform is switched off in the hippocampus and is detectable in the cortex. In addition, the ratio of another short RNA isoform (htra2-beta2) to htra2-beta1 is changed. Htra2-beta2 is not translated into protein, and probably helps to regulate the relative amounts of htra2-beta1 to beta3. We also observe activity-dependent changes in alternative splicing of the clathrin light chain B, c-src and NMDAR1 genes, indicating that the coordinated change of alternative splicing patterns might contribute to molecular plasticity in the brain.

Inverse correlation between IgG-antihinge region and antierythrocyte autoantibody in chronic benign and malignant cold agglutination.

Previous reports provided evidence of an immunosuppressive role of natural anti-F(ab')2 antibodies. If suppressive anti-F(ab')2 antibodies also regulated the autoantibody production in cold agglutination, one would expect high titers of anti-F(ab')2 to be associated with low titers of cold agglutinins. Indeed, our previous studies revealed an inverse correlation between IgG-anti-F(ab')2 and cold agglutinins. Many previous experiments focused on anti-F(ab')2 of an antiidiotypic nature. Recent epitope mapping showed that anti-F(ab')2 of healthy persons is not an antiidiotype but recognizes a hinge region sequence. We attempted to answer the question whether this IgG-antihinge antibody is responsible for the previously described association between anti-F(ab')2 and cold agglutinins. IgG-antihinge and IgG-anti-F(ab')2 antibody was determined and statistically analyzed in the serum of 334 patients with cold agglutination. Our experiments revealed a strong correlation between the concentrations of antihinge and the previously described anti-F(ab')2 antibody. The anti-F(ab')2 activity was competitively inhibited by a synthetic hinge peptide. Moreover, patients with high antihinge titers had low cold agglutinin titers, and vice versa. A stratification according to cold agglutinin specificity and disease etiology showed that the inverse correlation is present only in anti-I and anti-i patients suffering from monoclonal B-lymphocyte proliferation. In conclusion, our results confirm the correlation previously described for anti-F(ab')2 antibody and antierythrocyte autoantibody and define for the first time an association between an idiotype-independent anti-IgG autoantibody and cold agglutinin.

Chalcogen-analogs of amino acids. Their use in X-ray crystallographic and folding studies of peptides and proteins.

Using methionine-auxotrophic Escherichia coli strains quantitative biosynthetic replacement of the methionine residues by seleno- and telluromethionine but not by methoxinine was achieved in various model proteins, clearly indicating a limited tolerance in the editing range of methionyl-tRNA synthetase. For expression of the protein variants the acetyl derivatives of the chalcogen-analogs of methionine, obtained by a new and highly efficient synthetic procedure, proved to be the ideal source in the growth media as they were found to be significantly more stable than the underivatized methionine analogs. The conformational properties in solution, the folding and unfolding parameters as well as X-ray crystallographic data confirmed the highly isomorphous character of the atomic mutants and thus the usefulness of this concept in X-ray analysis of proteins. Quantitative replacement of cysteine residues by selenocysteine has recently been achieved using cysteine-auxotrophic E. coli strains, but a selective replacement of cysteine residues by employing the natural translational machinery of selenocysteine is also conceivable. We have therefore performed a detailed study on synthetic selenocysteine-peptides in order to determine the redox potential of this cysteine analog, and thus the ability of related peptide and protein analogs to undergo the correct oxidative folding. Since the redox potential of selenocysteine was found to be significantly more reducing than that of the parent amino acid, selective formation of a diselenide bridge in presence of additional cysteine residues is highly favored as well documented in the case of the synthetic bis-selenocysteine-endothelin I analog. These results confirm that even cysteine residues may represent an interesting target for the design and expression of isomorphous heteroatomic analogs of proteins.

A natural IgA-anti-F(ab')2gamma autoantibody occurring in healthy individuals and kidney graft recipients recognizes an IgG1 hinge region epitope.

Natural anti-IgG autoantibodies are found both in healthy individuals and in patients with certain diseases. One group of these Abs recognizes epitopes located in the F(ab')2 region of the IgG molecule. The immunoregulatory role of these Abs in healthy individuals, graft rejection, and disease was previously studied, usually with a focus on the characterization of anti-idiotypic Abs. In the present study, we characterize the epitope recognized by an anti-F(ab')2gamma autoantibody of the IgA isotype, which occurs in the serum of healthy individuals and kidney transplant recipients. The autoantibody described herein reacts strongly with F(ab')2gamma but only poorly with Fab(gamma) fragments, a binding pattern pointing to an epitope located in the hinge region. Using synthetic peptides, we identified a conformational epitope that overlaps the middle and part of the lower hinge region. Structural analyses of peptide constructs showed that a defined conformation of the first three residues of the lower hinge is required for a full expression of the epitope. Binding of IgA to the hinge region of IgG1 covers part of the physiologically active Fc domain, immobilizes the Fab arms, and thereby can be expected to exert immunoregulatory functions.

Cysteine racemization in peptide synthesis: a new and easy detection method.

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris-alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine-peptides. Baseline enantiomeric resolution of the D,L-S-beta-(4-pyridylethyl)cysteine, and thus quantification of D-enantiomer contents at levels < or = 1%, is easily achieved by capillary zone electrophoresis exploiting the host-guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.

Oxidative folding of cystine-rich peptides vs regioselective cysteine pairing strategies.

The methodology of regioselective cysteine pairings in synthetic multiple-cystine peptides has progressed in the past years to an efficiency that allows for at least three specific inter- and intrachain disulfide bridgings. Conformational studies on various multiple-cystine peptides like hormones, protease inhibitors, and toxins revealed that these bioactive peptides, generated by posttranslational processing of precursor proteins, are folded into miniprotein-like compact globular structures of remarkable stability. This strongly suggests protein domain or subdomain properties of these families of peptides, and thus sufficient sequence-encoded information for correct oxidative refolding under appropriate experimental conditions. From intensive research on the mechanisms and pathways of oxidative refolding of proteins in vivo and in vitro, the efficient methods have emerged for simulating nature in the regeneration of native folds not only for intact proteins, but also for protein domains and subdomains. In fact, the results obtained in the oxidative folding of excised protein fragments and of relatively low mass products of posttranslational processings show that this procedure is indeed a simple way of preparing peptides with several disulfide bonds, if optimization of reaction conditions is performed in terms of redox buffer, temperature, and additives capable of disrupting aggregates and of stabilizing nascent secondary structures. Moreover, with increased knowledge about stable, small natural cystine frameworks, their use instead of artificial templates should facilitate engineering of synthetic miniproteins with specific conformation and tailored functions.

Redox-active bis-cysteinyl peptides. I. Synthesis of cyclic cystinyl peptides by conventional methods in solution and on solid supports.

Cyclic mono-cystinyl active-site fragments of thioredoxin and thioredoxin reductase were synthesized as N-acetyl and C-amide octapeptides by conventional methods of peptide synthesis in solution and on solid supports. Using a side-chain protection based on acid-labile tert-butanol-derived groups and on the S-tert-butylthio unsymmetric disulfide for the thiol functions, in combination with N alpha-Z- or N alpha-Nps derivatives in the chain elongation steps, the synthesis in solution was carried out in straightforward manner yielding the fully protected octapeptides as well characterized compounds. Upon deprotection with trifluoroacetic acid and reduction of the unsymmetrical disulfides with tri-butylphosphine, the resulting bis-cysteinyl-octapeptides were oxidized in dimethylformamide with azodicarboxylic acid di-tert-butyl ester to produce the desired cyclic compounds in good overall yields. For the synthesis on solid supports a similar acid-labile side-chain protection was applied in combination with the N alpha 9-flourenylmethyoxycarbonyl derivatives in the chain elongation steps. Thereby acylations were performed with the related amino acid N-carboxyanhydrides (UNCAs) or by the O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetraf luoroborate/1- hydroxybenzotriazole (TBTU/HOBt) procedure. The solid phase synthesis of the two octapeptides led to unexpected difficulties in terms of recovery of peptidic material from the resins in the final acidolytic cleavage step as well as of racemization at the level of the cysteine residues by the TBTU/HOBt coupling method. Racemization was efficiently suppressed by employing the related pentafluorophenyl ester and this method led to crude octapeptide products of a degree of purity comparable to those obtained by the synthesis in solution. However, the recovery of the peptides from the resin, i.e., irreversible reattachment of cleaved peptidic material via alkylation of various side-chain functions, could not be avoided even using the most efficient scavengers or their cocktails.

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation.

Bis(cysteinyl)octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10(-3) M concentration was found to be very similar, but opposite to that of the Kox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of beta-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (approximately 90%). Similarly, operating under denaturing conditions, i.e., in 6 M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of beta-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence.

Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases.

The active sites of thiol-protein oxidoreductases consist of the characteristic Cys-X-X-Cys motif, and the redox potentials of these enzymes reflect the propensity of the bis(cysteinyl) sequence portion for disulfide loop formation. Thereby, as is known from comparing the three-dimensional (3D) structures of thioredoxin and glutaredoxin in the reduced and oxidized state, reduction of the disulfide bond is accompanied by minimal perturbation of the backbone folding of the active sites. In order to estimate the sequence-dependent intrinsic free energy of formation of the active-site disulfide loops in oxidoreductases, synthetic fragments corresponding to the sequences 31-38, 10-17, 134-141, and 34-41 of thioredoxin, glutaredoxin, thioredoxin reductase, and protein disulfide isomerase (PDI), respectively, were analyzed for their tendency to form 14-membered rings. For this purpose thiol/disulfide exchange experiments, with glutathione as reference redox pair, were performed on the bis(cysteinyl) octapeptides. As the free energy of ring closure of linear peptides consists mainly of the free energy of formation of the disulfide loop with a defined geometry from a statistical ensemble of conformations of the bis(cysteinyl) peptides, the observed differences in the equilibrium constants, although relatively small (within a factor 10), suggest that sequence-dependent information for loop formation is retained in the excised active-site fragments. These inherent redox potentials are, however, significantly affected and/or amplified in the native proteins by the conformational restraints imposed by the "structural domains" on the "functional domains".

Synthesis of neo-glycosylated L-alanyl-D-isoglutamine derivatives as potential immunoadjuvants.

The synthesis is described for muramyl-dipeptide-related amphiphilic analogs, where 1,10-diaminodecane served as a lipophilic C-terminal linker for the preparation of a bidentated L-alanyl-D-isoglutamine derivative and incorporation of aldonic and uronic acids at the N-termini as hydrophilic cores. For the N-acylation steps via lactones, suitable conditions were elaborated to allow for the use of unprotected or minimally protected aldonic and uronic acid lactones, respectively. These procedures may represent a useful general approach for the synthesis of neoglycopeptides.