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1.
Structure-function analysis of tritrpticin analogs: potential relationships between antimicrobial activities, model membrane interactions, and their micelle-bound NMR structures
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Tritrpticin is a member of the cathelicidin family of antimicrobial peptides. Starting from its native sequence (VRRFPWWWPFLRR), eight synthetic peptide analogs were studied to investigate the roles of specific residues in its biological and structural properties. This included amidation of the C-terminus paired with substitutions of its cationic and Phe residues, as well as the Pro residues that are important for its two-turn micelle-bound structure. These analogs were determined to have a significant antimicrobial potency. In contrast, two other peptide analogs, those with the three Trp residues substituted with either Phe or Tyr residues are not highly membrane perturbing, as determined by leakage and flip-flop assays using fluorescence spectroscopy. Nevertheless the Phe analog has a high activity; this suggests an intracellular mechanism for antimicrobial activity that may be part of the overall mechanism of action of native tritrpticin as a complement to membrane perturbation. NMR experiments of these two Trp-substituted peptides showed the presence of multiple conformers. The structures of the six remaining Trp-containing analogs bound to dodecylphosphocholine micelles showed major, well-defined conformations. These peptides are membrane disruptive and show a wide range in hemolytic activity. Their micelle-bound structures either retain the typical turn-turn structure of native tritrpticin or have an extended alpha-helix. This work demonstrates that closely related antimicrobial peptides can often have remarkably altered properties with complex influences on their biological activities. 相似文献
2.
Skok MV Lykhmus OY Bobrovnik SO Solodova OV Tzartos SJ Tsouloufis T Vanderesse R Marraud M 《Ukrainski? biokhimicheski? zhurnal》2002,74(4):54-60
The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding. 相似文献
3.
We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues. 相似文献
4.
Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein 总被引:3,自引:0,他引:3
G Fassina P P Roller A D Olson S S Thorgeirsson J G Omichinski 《The Journal of biological chemistry》1989,264(19):11252-11257
Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract. 相似文献
5.
Induction of feline immunodeficiency virus-specific cytotoxic T cells in vivo with carrier-free synthetic peptide. 总被引:3,自引:3,他引:0
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J N Flynn C A Cannon J A Beatty M Mackett M A Rigby J C Neil C Jarrett 《Journal of virology》1994,68(9):5835-5844
The role of cellular immunity in the establishment and progression of immunosuppressive lentivirus infection remains equivocal. To develop a model system with which these aspects of the host immune response can be studied experimentally, we examined the response of cats to a hybrid peptide containing predicted T-and B-cell epitopes from the gag and env genes of feline immunodeficiency virus (FIV). Cats were immunized with an unmodified 17-residue peptide incorporating residues 196 to 208 (from gag capsid protein p24) and 395 to 398 (from env glycoprotein gp120) of the FIV Glasgow-8 strain by using Quil A as an adjuvant. Virus-specific lymphocytotoxicity was measured by chromium-51 release assays. The target cells were autologous or allogeneic skin fibroblasts either infected with recombinant FIV gag vaccinia virus or pulsed with FIV peptides. Effector cells were either fresh peripheral blood mononuclear cells or T-cell lines stimulated with FIV peptides in vitro. Cytotoxic effector cells from immunized cats lysed autologous, but not allogeneic, target cells when they were either infected with recombinant FIV gag vaccinia virus or pulsed with synthetic peptides comprising residues 196 to 205 or 200 to 208 plus 395. Depletion of CD8+ T cells, from the effector cell population abrogated the lymphocytotoxicity. Immunized cats developed an antibody response to the 17-residue peptide immunogen and to recombinant p24. However, no antibodies which recognized smaller constituent peptides could be detected. This response correlated with peptide-induced T-cell proliferation in vitro. This study demonstrates that cytotoxic T lymphocytes specific for FIV can be induced following immunization with an unmodified short synthetic peptide and defines a system in which the protective or pathological role of such responses can be examined. 相似文献
6.
The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability. Proteins 33:97–106, 1998. © 1998 Wiley-Liss, Inc. 相似文献
7.
Patricia Cano-Sanchez Beatrice Severino V V Sureshbabu Joe Russo Tatsuya Inui Fa-Xiang Ding Boris Arshava Jeff Becker Fred Naider 《Journal of peptide science》2006,12(12):808-822
Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N- and C-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lys(n) (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol-water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies. 相似文献
8.
The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn (34)). Using synthetic peptides, we have previously investigated the role of Asn (34) in the context of simple hydrophobic transmembrane peptides. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-Ala sequence (Leu 12) or Leu residues at the native positions found in PLB (Leu 9). Asn-containing Leu 9 and Leu 12 peptides were synthesized with a single Asn residue located either one amino acid (N+/-1) or one turn of the helix (N+/-4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle. 相似文献
9.
Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop
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The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor. 相似文献
10.
Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions. 相似文献
11.
An alpha-amylase inhibitor (0.53-inhibitor) was digested with pepsin and the cystine-containing peptides were isolated by SP-Sephadex C-25 column chromatography and high voltage paper electrophoresis. Amino acid compositions and partial sequences of these peptides and amino acid compositions of the peptides separated after performic acid oxidation revealed the presence of 4 disulfide bonds in the inhibitor. They were formed between residues 6 and 115, 20 and either 41 or 42, 99 and either 41 or 42, and 28 and 83. The disulfide bond partners of residues 20 and 99 were not conclusively determined because of the difficulty of cleavage of the Cys-Cys bond at residues 41 and 42. Among 9 cysteines in this inhibitor Cys-54 must be in a free form, since no evidence was obtained for its contribution to disulfide bond formation. 相似文献
12.
T Takahashi S Hiramoto S Wato T Nishimoto Y Wada K Nagai H Yamaguchi 《Journal of biochemistry》1999,126(5):838-844
Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue. 相似文献
13.
Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388–395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261–287) and cluster 3 (residues 147–151) peptides followed by cluster 2 (residues 290–302) peptide. A peptide corresponding to LPL cluster 4 (residues 405–414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications. 相似文献
14.
Primary structure of belladonna mottle virus coat protein 总被引:1,自引:0,他引:1
S Suryanarayana N A Rao M R Murthy H S Savithri 《The Journal of biological chemistry》1989,264(11):6273-6279
The coat protein of belladonna mottle virus (a tymovirus) was cleaved by trypsin and chymotrypsin, and the peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase, and ion pair chromatography. The peptides were sequenced manually using the 4-N, N-dimethylaminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double-coupling method. The chymotryptic peptides were aligned by overlapping sequences of tryptic peptides and by homology with another tymovirus, eggplant mosaic virus. The belladonna mottle virus is more closely related to eggplant mosaic virus than to turnip yellow mosaic virus, the type member of this group, as evident from the sequence homologies of 57 and 32%, respectively. The accumulation of basic residues at the amino terminus implicated in RNA-protein interactions in many spherical plant viruses was absent in all the three sequences. Interestingly, the amino-terminal region is the least conserved among the tymoviruses. The longest stretch of conserved sequence between belladonna mottle virus and eggplant mosaic virus was residues 34-44, whereas it was residues 96-102 in the case of belladonna mottle virus and turnip yellow mosaic virus. A tetrapeptide in the region (residues 154-157) was found to be common for all the three sequences. It is possible that these conserved regions (residues 34-44, 96-102, 154-157) are involved in either intersubunit or RNA-protein interactions. 相似文献
15.
Anish Suri Ilan Vidavsky Koen van der Drift Osami Kanagawa Michael L Gross Emil R Unanue 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(3):1235-1243
We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus. 相似文献
16.
Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1. 总被引:8,自引:0,他引:8
Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition. 相似文献
17.
The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif 总被引:3,自引:0,他引:3
K Vancompernolle J Vandekerckhove M R Bubb E D Korn 《The Journal of biological chemistry》1991,266(23):15427-15431
Actobindin is an 88-amino acid polypeptide, containing two almost identical repeated domains of 33 and 34 residues. Depending on the molar ratios in which they are mixed, actobindin binds either one or two actin molecules. We cross-linked actobindin and actin in the 1:1 complex, using the zero-length cross-linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. The cross-linked peptides were purified after consecutive CNBr cleavage and trypsin and Staphylococcus protease V8 digestions, and the cross-linked side chains were identified by amino acid sequencing. Isopeptide linkages were formed between residues Glu-100 of actin and Lys-16 of actobindin. In addition, we found a connection between one or more of the acidic residues 1,2, or 3 of actin and Lys-16 and Lys-52 of actobindin. The cross-linked regions in actobindin contain Leu-Lys-His-Ala-Glu-Thr motifs, similar to sequences observed in several other actin-binding proteins. 相似文献
18.
Fleckenstein B Qiao SW Larsen MR Jung G Roepstorff P Sollid LM 《The Journal of biological chemistry》2004,279(17):17607-17616
Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease. 相似文献
19.
N Miyoshi T Yonemochi S Tomono R Fukutomi Y Nakamura H Ohshima 《Analytical biochemistry》2012,429(2):124-131
In this study, we have developed a novel method to identify isothiocyanate (ITC)-targeted molecules using two well-studied ITCs: benzyl ITC (BITC) and phenethyl ITC (PEITC). The principle of this method is based on identifying a pattern of differences between BITC and PEITC given that they show similar chemical and biological behaviors. For method validation, dithiothreitol-reduced bovine insulin as a model molecule was incubated with either BITC or PEITC, and digested peptides were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS). Three peptides-NYCN, FVNQHLCGSHLVE, and ALYLVCGE-were identified as being adducted with BITC or PEITC on their cysteine residues. Each set of peptides adducted with either BITC or PEITC showed retention times (RT(BITC)
20.
Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides. 总被引:1,自引:0,他引:1
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To study the structure relationship of different Tat domains, the full-length Tat protein Tat1-86, the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat1-86 and Tat1-72 transactivated beta-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat1-86 and Tat1-72 with the sulfhydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 and Tat38-60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation. 相似文献