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A replacement of both Cys residues by His in oxytocin (OXT) sequence allows for the formation of the stable complex with the {NH(2), N(Im), N(Im(macrochelate))} binding mode at the physiological pH. The detailed potentiometric and spectroscopic studies on the Cu(II) complexes of [His(1,6)]OXT, together with high resolution NMR investigations on 3D structures of Cu(II) complexes with [His(1,6)]OXT and [His(1,6)]AVP analogues are presented and discussed. Exchange of the Cys-S-S-Cys bridge by the His-Cu(II)-His motif is very promising, because the resulting complexes retain topological similarity to the native S-S bridged AVP and OXT at pH values corresponding to the physiological pH.

We have characterized the interaction between the Zn(2+) ions and the histidine analogues of oxytocin and arginine-vasopressin. Potentiometric methods were used for the determination of the stoichiometry of the complexes formed and the calculation of their stability constants. The NMR measurements revealed detailed structures of the complexes and confirmed the binding mode at physiological pH.

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

Recently, a third evolutionarily conserved gene, NWC, was discovered within the recombination activating gene (RAG) locus, known to contain the RAG1 and RAG2 genes. Here, we identify and characterize the murine endogenous NWC protein which has no homology to any known protein and is ubiquitously expressed. In the cell, the NWC protein which has been suggested to function as a transcriptional repressor, is found in the cytoplasm as well as in the nucleus.

A new vasopressin analogue, [His1,6]AVP, was synthesized and characterized by potentiometric measurements as well as by UV-Vis, CD and EPR spectroscopy. At the physiological pH the peptide forms a stable complex with Cu2+ ions which is characterized by the {NH2, NIm, NIm(macrochelate)} binding mode. The replacement of both Cys by His residues in the vasopressin sequence results in a very significant increase in the efficiency of Cu2+ binding.

NWC, third evolutionarily conserved gene within RAG locus is transcribed at high level in all cells except mature T and B lymphocytes and their RAG negative progenitors. It is so, because in lymphocytes expression of NWC is regulated by RAG-1 promoter, while in other cells it is controlled by RAG-2 intragenic promoter which in T and B lymphocytes is silent. Here we show that lymphocyte-specific inactivation of NWC promoter is caused by CpG island hypermethylation accompanied by site-specific blocking of chromatin accessibility, which in contrast to RAG promoters, is not accompanied by expected posttranslational modifications of histone H3. These results indicate that accessibility of NWC promoter and RAG promoters to trans-acting factors is regulated by different epigenetic mechanisms. The implications of our findings for understanding mechanisms regulating transcription within RAG/NWC locus in different cells are discussed and the model of epigenetic control of this locus is proposed.

Molecular cloning of targeting vectors (TgVs) is a prerequisite procedure for gene disruption in embryonic stem cells. In cases where target genes display complex features (e.g., gene overlap, alternative exon usage), TgVs must mediate deletions with very high precision to prevent unwanted effects. This is often difficult to achieve by procedures using restriction endonucleases and DNA ligases. Therefore, to prepare TgVs for inactivation of two complex genes of immunological interest: PTPRF and NWC, we employed an alternative method, which involves engineering bacterial artificial chromosomes (BACs) by inducible, plasmid encoded "Red/ET recombinase" expression system. Here, we report rapid and efficient construction of PTPRF and NWC TgVs without using restriction endonucleases.

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.

This paper describes the first potent and selective p300 histone acetyltransferase (HAT) inhibitor which is effective in live cells. This compound 7 is a coenzyme A analogue conjugated to a cell permeabilizing oligoArg peptide via disulfide linkage. This compound was shown to block cellular histone acetylation and transcription using a p300-sensitive reporter. It should thus be broadly useful for dissecting the role of p300 HAT activity in physiologic and disease states.

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.

Recombination-activating gene (RAG)1 and RAG2 encode T and B lymphocyte-specific endonucleases indispensable for rearrangements of antigen-receptor gene segments but also capable of causing deleterious chromosome rearrangements. The mechanisms regulating RAG expression and repression are not clear. Here we identify NWC, a third evolutionarily conserved gene within the RAG locus, and show that it is ubiquitously expressed, with the notable exception of RAG-nonexpressing immature and mature T and B lymphocytes because in lymphocytes it is regulated by the RAG1 promoter and transcribed as RAG1-NWC hybrid mRNA molecules. We also show that in all other cells NWC is controlled by the RAG2 intragenic promoter, which in immature and mature T and B lymphocytes is silent. The possible implications of these findings for understanding the activation and inactivation of RAG genes in lymphocytes and their repression in other cells are discussed.

The review presents three hypotheses concerning the amino acid complementarity: 1) the Mekler-Blalock antisense hypothesis; 2) the Root-Bernstein approach based on stereochemical complementarity of amino acids and anti-amino acids coded by anticodons read in parallel with the coding DNA strand; 3) Siemion hypothesis resulting from the periodicity of the genetic code. The current state of knowledge as well as the results of the implementations of these hypotheses are compared. A special attention is given to Root-Bernstein and Siemion hypotheses, which differ in only few points of the complementarity prediction. We describe methods of investigation of peptide-antipeptide pairing, including circular dichroism, mass spectrometry, affinity chromatography and other techniques. The biological applications of complementarity principle are considered, such as search for bioeffector-bioreceptor interaction systems, the influence of peptide-antipeptide pairing on the activity of peptide hormones, and the application of antipeptides in immunochemistry. The possible role of amino acid-anti-amino acid interactions in the formation of the spatial structures of peptides, proteins and protein complexes is discussed. Such problems as the pairing preferences of protein-protein interfaces, the role of the pairing in the creation of disulfide bonds and the possible appearance of such interactions in beta-structure are also examined. The main intention of the paper is to bring the complementarity problem to the attention of the scientific community, as a possible tool in proteomics, molecular design and molecular recognition.

Using cDNA-Representational Difference Analysis it was found that expression of Opg, Ctse, Krt2-4, Fut-2, 24p3 and Wif-1 genes was elevated in intestinal adenomas as compared to normal epithelial cells of Apc(Min/+) mutant mice. Expression of Wif-1, which encodes Wnt inhibitory factor-1 was also detected in a number of tumor cell lines of epithelial cell origin including two human colon adenocarcinoma cell lines. The possible role of Wif-1 over-expression in the etiology of colorectal cancer is discussed.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. Antiviral drugs targeted against lytic viral replication have limited efficacy in these disease settings. EBV infection of peripheral blood mononuclear cells induces growth proliferation and the EBV latency Epstein-Barr virus-encoded nuclear antigen (EBNA)2 transcriptional transactivator (TAT) is essential for this response. EBNA2 targets the cellular DNA-binding protein CBF1 to mimic activated Notch signaling. A 10-aa peptide from the CBF1 interaction domain of EBNA2 was synthesized as a fusion with the protein transduction domain of HIV-1 TAT. The EBNA2-TAT peptide blocked EBNA2-CBF1 interaction in an in vitro GST affinity assay and labeling with fluorescein confirmed that the EBNA2-TAT peptide efficiently entered cultured B cells. Neither EBNA2-TAT, nor a mutant peptide with a 2-aa substitution that was unable to block the EBNA2-CBF1 interaction, significantly affected the growth of non-EBNA2-expressing EBV(-) B cells or Burkitt's lymphoma Akata cells. However, treatment of an EBV-immortalized lymphoblastoid cell line with the EBNA2-TAT peptide stopped cell growth and reduced cell viability. RT-PCR analyses of gene expression in the peptide-treated lymphoblastoid cell line cultures revealed that EBNA2-TAT treatment down-regulated the EBNA2-responsive viral LMP1 and LMP2 genes and cellular CD23, intercellular adhesion molecule 1, BATF, and Cdk1 genes while up-regulating expression of the cyclin-dependent kinase inhibitor p21. EBV-induced outgrowth of B cells from cultured peripheral blood mononuclear cells was also blocked in a dose-responsive manner by the EBNA2-TAT peptide. This study suggests that cell-permeable EBNA2 peptides may have potential as novel anti-EBV therapeutics.

Interleukin-1 receptor antagonist (IL-1Ra) and vaccinia virus protein C10L share a VTXFYF motif, with X being Lys or Arg residue, respectively. Peptides of such sequence compete successfully with IL-1 for the cellular receptor. A pair of complementary peptides, based on the Siemion's hypothesis on the periodicity of the genetic code (QWLNIN and QWANIN), and another pair, in which, following the Root- Bernstein theory, Lys was used as complementary amino acid to Phe (QWLKIK and QWAKIK), were investigated for the peptide-antipeptide interactions using mass spectrometry (ESI-MS) and circular dichroism (CD) methods. The CD measurements indicated some conformational changes, more pronounced in the Siemion's pairs, however, no heterodimer formation was found by MS. In the region of IL-1 receptor situated close to the position of IL-1Ra in the IL-1Ra-receptor complex, a KQKL motif is present, suggesting a possibility of complementary recognition of the Root-Bernstein type in the IL-1 receptor. The biological activity of the complementary peptides is similar to that of the original ones. They efficiently compete with IL-1 and show moderate immunosuppressory activity in humoral and cellular immune response. The inhibition of the IL-1-IL-1 receptor interaction may result from the complementary peptides acting as mini-receptors with affinity for IL-1.

Lys-CoA (1) is a selective inhibitor of p300 histone acetyltransferase (HAT) but shows poor pharmacokinetic properties because of its multiply charged phosphates. In an effort to overcome this limitation, truncated derivatives of 1 were designed, synthesized and tested as p300HAT inhibitors as well as substrates for the CoA biosynthetic bifunctional enzyme phosphopantetheine adenylyltransferase-dephospho-CoA kinase (PPAT/DPCK). Lys-pantetheine (3) and Lys-phosphopantetheine (2) showed no detectable p300HAT inhibition whereas 3'-dephospho-Lys-CoA (5) was a modest p300 inhibitor with IC(50) of 1.6 microM (compared to IC(50) of approximately 50 nM for 1 blocking p300). Compound 2 was shown to be an efficient substrate for PPAT whereas 5 was a very poor DPCK substrate. Further analysis with 3'-dephospho-Me-SCoA (7) indicated that DPCK shows relatively narrow capacity to accept substrates with sulfur substitution. While these results suggest that truncated derivatives of 1 will be of limited value as lead agents for p300 blockade in vivo, they augur well for prodrug versions of CoA analogues that do not require 3'-phosphate substitution for efficient binding to their targets, such as the GCN-5 related N-acetyltransferases.

Histone acetyltransferases (HATs) use acetyl CoA to acetylate target lysine residues within histones and other transcription factors, such as the p53 tumor suppressor, to promote gene activation. HAT enzymes fall into subfamilies with divergence in sequence and substrate preference. Several HAT proteins have been implicated in human cancer. We have previously reported on the preparation of peptide-CoA conjugate inhibitors with distinct specificities for the p300/CBP [cAMP response element binding protein (CREB)-binding protein] or GCN5 HAT subfamilies. Here we report on the crystal structure of the GCN5 HAT bound to a peptide-CoA conjugate containing CoA covalently attached through an isopropionyl linker to Lys-14 of a 20-aa N-terminal fragment of histone H3. Surprisingly, the structure reveals that the H3 portion of the inhibitor is bound outside of the binding site for the histone substrate and that only five of the 20 aa residues of the inhibitor are ordered. Rearrangements within the C-terminal region of the GCN5 protein appear to mediate this peptide displacement. Mutational and enzymatic data support the hypothesis that the observed structure corresponds to a late catalytic intermediate. The structure also provides a structural scaffold for the design of HAT-specific inhibitors that may have therapeutic applications for the treatment of HAT-mediated cancers.

In transcription assays, Renilla luciferase-expressing plasmids (more specifically pRL-TK) are commonly used as an internal control of transfection efficiency. Normalization of the experimental reporter gene transcription to the internal control reporter gene transcription minimizes variability of obtained results caused by differences in transfection efficiency between different samples of transfected cells. It is obvious that co-transfection with other plasmids or applied treatments should not affect the activity of the control reporter. Here we report that expression of the control Renilla luciferase encoded by pRL-TK plasmid was enhanced by co-transfection with vectors expressing orphan nuclear receptors Nur77 family (Nur77, Nurr1, Nor-1), leading to misinterpretation of the assay results. Further, we show that for Nurr1, phRG-B (a promoterless reporter plasmid containing synthetic Renilla luciferase gene) is a better control reporter vector than HSV-TK containing vectors. Finally, we noted the lack of effect of Nurr1 protein on the Fas Ligand promoter-driven transcription.

It was previously reported that thymic lymphomas of anti HY-TCR transgenic mice were resistant to ionomycin but were sensitive to 50 microM of etoposide. We selected lymphoma clones with strong expression of anti-apoptotic proteins Bcl-2 or Bcl-xL and found that in contrast to proteins encoded by exogenous genes in other model systems, over-expression of endogenous Bcl-2 and Bcl-xL proteins failed to protect against etoposide-induced apoptosis. Susceptibility of lymphoma cells was not diminished even to suboptimal concentration of 0.5 microM of etoposide. Treatment with etoposide did not decrease the ratio of anti-apoptotic Bcl-2 and Bcl-xL proteins to pro-apoptotic Bax protein. Results presented here suggest that high expression of Bcl-2 and Bcl-xL may not diminish susceptibility of T-cell lymphomas to chemotherapy with etoposide.

In this paper we present recent data on molecular function of APC protein and Wnt signaling pathway. Role of a link between APC and Wnt signaling in the etiology of colorectal cancer as well as significance of a mutant APC mice as a model of cancer in man are discussed.

Cyclolinopeptide A (CLA) is an immunosuppressive peptide of the sequence c-(-Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), isolated from linseed. Since another cyclic, hydrophobic, immunosuppressive peptide, cyclosporin A, has potent antimalarial activity, CLA and a series of its analogues were synthesized on solid phase and tested for inhibition of the human malarial parasite Plasmodium falciparum in culture. The results were compared with the influence of these agents on humoral and cellular immune responses. There was no clear correlation between the structure of the peptides, their immunosuppressive activity, and their antimalarial activity. However, the antimalarial activity of the peptides was apparently connected with the strong hydrophobic nature of CLA. Substitution of a less hydrophobic residue into the peptide chain led to a decrease in or even loss of detectable activity, although such peptides retained the immunosuppressive properties. A possible explanation is that the antimalarial effect of CLA and analogues may result from their influence on cell membranes rather than on some specific receptor such as cyclophilin. In agreement with this idea, binding of CLA to purified P. falciparum cyclophilin was not detected except at very high concentrations. Substitution of D-aromatic residues into the CLA molecule led to a decrease in immunosuppressive activity but had little effect on antimalarial activity, which for these peptides was of the same order as for CLA. We have therefore demonstrated that the cyclolinopeptides are a class of compound not previously shown to have antimalarial activity, and that in a series of analogues there was no correlation between antimalarial and immunosuppressive effects.

The results of the investigation of immunosuppressive activity of cyclolinopeptide A (CLA--cyclic hydrophobic nonapeptide present in the linseeds) and its analogs are discussed. The results obtained for other natural cyclic peptides showing structural similarities with CLA (antamanide, cycloamanides, hymenistatin, hymenamides) are also reviewed. It results from these investigations that the molecular mechanism of the CLA action is the same as that of cyclosporin A and FK-506 compound, i.e. it consists in formation of the complex with cyclophilin and inhibition--in this form--of the phosphatase activity of calcineurin. The results also suggest that the immunosuppressive activity of these compounds resides in their--Pro-Xxx-Phe- fragment, where Xxx is a hydrophobic (e.g. Leu, Val) or aromatic amino acid residue.

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium-dependent, but not calcium-independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl-prolyl cis-trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.

Linear and cyclic analogues of cyclolinopeptide A (CLA) in which one or both phenylalanine residues in fragment Pro6-Pro7-Phe8-Phe9 were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogues retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analogue modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established.

We measured 1H-NMR, fluorescence and CD spectra of cyclolinopeptide A (CLA), its tyrosine analogues with each or both phenylalanines substituted by tyrosine (c-[LeuIleIleLeuValProProTyrPhe], c-[LeuIleIleLeuValProProPheTyr] and c-[LeuIleIleLeuValProProTyrTyr]), and their linear counterparts with the starting sequence leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe (LA). It follows from CD spectra that the conformations of all cyclic peptides are similar to that of CLA; the conformations of linear peptides are more diversified, with the conformation of [Tyr9]LA being most similar to CLA. NMR studies suggest that aromatic rings in cyclic peptides are situated perpendicular to each other, manifesting edge-to-face pairing. Accordingly, the residue in position 9 is shielded ('edge'), and a residue in position 8 is the shielding one ('face'). This effect is not present in the case of linear peptides. Fluorescence quantum yields were much lower for cyclic peptides than for linear ones, indicating the interaction of closely located aromatic chromophores. Those quantum yields depend on the relative position of Tyr in the peptide chain. Another factor influenced by the position in the peptide chain is the optical activity of aromatic side chains (optically active in position 8, inactive in position 9). This phenomenon could be explained by the differences in the side-chain conformation of both aromatic residues.

We demonstrated that a linear peptide Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe, the analog of a cyclic nonapeptide c-(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe) possesses a distinct immunosuppressive activity. We synthesized a series of eight other analogs of the linear peptide. The series consists of the nonapeptides protected on N- and/or C-terminus by acetyl-, succinyl-, and amido- functions and of Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe elongated on N- and/or C-terminus by additional glycine residues. It was found that the protection of the terminal functional groups of the peptide slightly increases its immunosuppressor potency. At the same time the elongation of the peptide chain on both termini by additional glycine residues evokes a distinct increase of the immunosuppressive activity.