Functional genetics-directed identification of novel pharmacological inhibitors of FAS- and TNF-dependent apoptosis that protect mice from acute liver failure.

shRNA-mediated gene-silencing technology paired with cell-based functional readouts reveals potential targets directly, providing an opportunity to identify drugs against the target without knowing the precise role of the target in the pathophysiological processes of interest. By screening a lentiviral shRNA library targeting for major components of human signaling pathways and known drug targets, we identified and validated both canonical as well as 52 novel mediators of FAS and TNF ligand-induced apoptosis. Presence of potential therapeutic targets among these mediators was confirmed by demonstration of in vivo activity of siRNAs against four identified target candidates that protected mice from acute liver failure (ALF), a life-threatening disease with known involvement of death receptor (DR)-mediated apoptosis. Network-based modeling was used to predict small-molecule inhibitors for several candidate apoptosis mediators, including somatostatin receptor 5 (SSTR5) and a regulatory subunit of PP2A phosphatase, PPP2R5A. Remarkably, pharmacological inhibition of either SSTR5 or PPP2R5A reduced apoptosis induced by either FASL or TNF in cultured cells and dramatically improved survival in several mouse models of ALF. These results demonstrate the utility of loss-of-function genetic screens and network-based drug-repositioning methods for expedited identification of targeted drug candidates and revealed pharmacological agents potentially suitable for treatment of DR-mediated pathologies.

Mutations in fd phage major coat protein modulate affinity of the displayed peptide.

Multibillion-clone libraries of phages displaying guest peptides fused to the major coat protein pVIII (landscape libraries) are a rich source of probes for proteinaceous and non-proteinaceous targets. As opposed to the pIII-type fusion phages, which display peptides as independent structural domains, the guest peptides in the pVIII-fusion phages can be structurally and functionally influenced by contiguous subunits. To decipher the impact of the locale of a guest peptide on its affinity characteristics, we constructed a library of phages carrying beta-galactosidase-binding peptide ADTFAKSMQ at the N-terminus of the pVIII protein surrounded by random amino acids. It was found that mutagenesis of amino acids 12-19 (domain C) has polar effects on target binding affinity of the displayed peptide. The phages with highest affinity are characterized by: (i) a net electrostatic charge around -1 of domain C of the mutated phages at pH 7.0; (ii) a lower radius of cylinder coaxial to alpha-helix formed by domain C; (iii) a lower higher occupied molecular orbital (HOMO) of domain C leading to a decreased formation of hydrogen bonds and (iv) positively charged surface and torsion energy of domain C, which may require a conformational transition of N-terminal peptide ADTFAKSMQ for its binding with beta-galactosidase. Influence of the guest peptide on the diversity of mutations in the neighboring landscape area was also observed.

[Construction of original artificial immunogens--candidate vaccines against HIV-1]. / Konstruirovanie original'nykh iskusstvennykh immunogenov--kandidatov v vaktsiny protiv HIV-1.

An original method for making effective artificial vaccines has been developed. Immunogens are virus-like particles containing a DNA molecule covered with recombinant proteins carrying the pathogen epitopes. The recombinant proteins are exposed on the surface of the particle and are attached to DNA via spermidine-polygluquine-glutathione or galactopyranoside conjugates (depending on the hybrid protein composition). Two variants of artificial immunogens-candidates for antiHIV vaccine have been prepared.

Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble.

Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high beta-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given.

Conserved transactivation domain shared by interferon regulatory factors and Smad morphogens.

Interferon regulatory factors (IRFs) regulate the transcription of both interferon-inducible genes and interferons themselves. Along with the N-terminal, DNA-binding, winged-helix domain, most IRFs contain the C-terminal domains that are shown to be related to the C-terminal domains in the proteins of the Smad family that mediate transcription activation in the transforming growth factor response pathway. Comparison of the IRF-Smad alignment to the known three-dimensional structure of human tumor suppressor Smad4 suggests that a conserved loop, equivalent to Loop 3 in Smad 4, is a determinant of protein-protein interaction in IRFs.

ProMSED: protein multiple sequence editor for Windows 3.11/95.

MOTIVATION: Most protein sequence alignment algorithms give similar results on closely related proteins, while manual intervention may be needed for distantly related molecules. To correct the alignment, it is often necessary to repeat calculations on selected parts of the alignments and edit the alignment manually. Software implementing such interactive alignment procedures is of significance. RESULTS: This paper presents a new MS Windows application called ProMSED for both automatic and manual protein sequence alignment. The program reads main sequence formats and has a user-friendly interface. ProMSED performs automatic (ClustalV algorithm) alignments, alignment visualization and editing, and it allows sequences to be aligned interactively leaving previously aligned regions unchanged. Manual alignment and sequence analysis are facilitated by colouring schemes reflecting amino acid similarity of mutational and physicochemical properties. The interactive alignment of a diverged set of reverse transcriptases has located four out of six known conserved motifs. AVAILABILITY: ProMSED is available on request from the authors. DEMO is available from ftp://ftp.ebi.ac.uk/pub/ software/dos/promsed/ or ftp://iubio.bio.indiana.edu/molbio/ ibmpc/.

[Use of a phage peptide library in mapping the group-specific hemagglutinating domain of alpha-virus glycoprotein E2]. / Ispol'zovanie fagovoi biblioteki peptidov v kartirovanii gruppospetsificheskogo gemaggliutiniruiushchego domena glikoproteina E2 al'fa-virusa.

Phage display peptide library f88-4/15 (G. P. Smith, USA) was used for mapping the hemagglutination activity domain of glycoprotein E2 of alphaviruses. Using affinity selection and ELISA, we selected the clones binding monoclonal antibody 4H5 to Venezuelan equine encephalomyelitis virus and inhibiting alphavirus hemagglutinating activity. Analysis of the similarity between the peptides amino acid sequences with the alphavirus glycoprotein E2 sequences revealed a structural motive of 4 amino acid residues (HTSR) which was identified in the 85-88 region. Bacteriophages F36 and F19 contained motives corresponding to 102-SXXM-105 and 109-AXXP-112 regions in alphavirus proteins E2. These data permit us to propose that the detected regions are fragments of a group-specific alphavirus hemagglutination domain.

[Cloning proviral DNA sequences of the human T-cell leukemia virus (HTLV-1) from the genome of cultured MT-4 cells]. / Klornirovanie provirusnykh DNK-posledovatel'nostei virusa T-kletochnogo leikoza cheloveka tipa 1 (HTLV-1) iz genoma kul'tury kletok MT-4.

MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles. A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction. Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined. The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges. Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).

Design of immunogens as components of a new generation of molecular vaccines.

Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.

Design of four-helix bundle protein as a candidate for HIV vaccine.

To be efficient, a synthetic vaccine should contain different T and B cell epitopes of human immunodeficiency virus (HIV) antigens, and the B epitope regions in the vaccine and in the HIV should be conformationally similar. We have suggested previously the construction of vaccines in the form of a protein with a predetermined tertiary structure, namely a four-alpha-helix bundle. Antigenic determinants of cellular and humoral immunity are blocks for the vaccine design. From experimentally studied HIV-1 T and B cell epitopes, we constructed a sequence of a four-helix protein TBI (T and B cell epitopes containing immunogen). The gene of the protein was synthesized and the protein was produced in C600 Escherichia coli cells under recA promoter from Proteus mirabelis. CD spectroscopy of the protein demonstrated that 30% of amino acid residues adopt an alpha-helical conformation. Mice immunized with TBI have shown both humoral and cellular immune responses to HIV-1. The obtained data show that the design of TBI was successful. The synthesized gene structure makes possible further reconstruction and improvement of the protein vaccine structure.

PROANAL version 2: multifunctional program for analysis of multiple protein sequence alignments and for studying the structure--activity relationships in protein families.

A new version of the program PROANAL is described. A multiple linear regression analysis of the protein structure--activity relationship allows one to investigate the combinations of protein sites and factors influencing the activity. The program also provides the possibility to seek out protein sites, conservative or variable in variations of physicochemical characteristics, and regions with high or low values of these characteristics. PROANAL2 may be useful in the simulation of protein-engineering experiments and in the search of a number of protein regions such as functional sites, secondary structures, solvent-exposed regions, T- and B-cell antigenic determinants, etc.

[Preparation of a mutant human tumor necrosis factor with increased resistance to proteases]. / Poluchenie mutantnogo faktora nekroza opukholei cheloveka s povyshennoi ustoichivost'iu k proteazam.

The potential proteolysis sites of human TNF are considered. By site-directed mutagenesis the Arg-31 residue of mature TNF was substituted by Gln. The analysis of cytotoxicity of initial and mutant (R31Q) proteins on mouse L929 fibroblasts did not reveal any differences in biological activity. For the mutant protein a change in proteolysis dynamics was shown in contrast to the natural variant: mutant TNF displayed increased stability when treated with trypsin.

Artificial protein vaccines with predetermined tertiary structure: application to anti-HIV-1 vaccine design.

A successful approach to the development of a safe and effective synthetic vaccine requires that different B and T cell epitopes of the infectious agent be included in the vaccine construction. In this paper we suggest a new approach to vaccine design in the form of an artificial protein with a predetermined tertiary structure (PTS vaccines). Based on B and T cell epitope properties, we substantiate the possible use for vaccine construction of one well-known protein spatial motif--the four-alpha-helix bundle. Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) are used as blocks for vaccine design. General principles of PTS vaccine construction have been applied to anti-HIV-1 vaccine design.

Algorithm and computer program Pro__Anal for analysis of relationship between structure and activity in a family of proteins or peptides.

In this paper we introduce a computer algorithm and program Pro__Anal for analysis of the structure-activity relationship in a family of evolutionarily related (and/or artificially mutated) proteins/peptides. The program uses aligned amino acid sequences with data of their activity (pK, Km, ED50 or any other) and searches for correlations between data on activity and various physico-chemical characteristics of different regions in primary structures. In automatic mode, the program generates and verifies hypotheses on the disposition of a sequential modulating region in a protein, and key characteristics of the region. In manual mode, users can generate and analyze their own hypotheses. The program is implemented on IBM PC or compatible computers. It is designed to be easily handled by the occasional computer user and yet it is powerful enough for experienced professionals. Pro__Anal operation is demonstrated on the example of finding modulating centers in a family of disintegrins-proteins from snake venoms which inhibit fibrinogen interaction with platelet receptors. In another example it is shown that the immunogenicity of peptides is connected with their positive charge.

[Design of four-helix protein--a possible vaccine against human immunodeficiency virus (HIV-1)]. / Konstruirovanie chetyrekhspiral'nogo belka--vozmozhnoi vaktsiny protiv virusa immunodefitsita cheloveka (HIV-1).

Successful approach to the development of safe and effective synthetic vaccines requires that different B- and T-cell epitopes of the infectious agent be included into the vaccine construction. It is suggested that vaccines should be constructed as proteins with both optimal epitope composition and predetermined tertiary structure. Based on analysis of B-cell and T-cell epitope properties, a possibility to use one well-known protein spatial motif--four-alpha-helix bundle--for vaccine construction is substantiated. Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) can be used as blocks for vaccine design. Nonloop B-epitopes and nonhelical T-epitopes may be introduced in the protein N- and C-terminal regions. General principles of PTS-vaccine construction have been applied to anti-HIV-1 vaccine design. Experimentally studied T- and neutralizing B-cell epitopes from HIV-1 proteins were analyzed. The sequence of one possible four-alpha-helix protein vaccine has been constructed. Predicted secondary structure and T- and B-cell epitopes of this protein coincided with the planned ones. The amino acid composition of the protein was found to be consistent with the composition of globular water-soluble proteins. The gene of the protein with codon composition optimal for expression in E. coli has been synthesized. The advantages and limitations of this approach to vaccine design are discussed.

Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.