N. V. Maluchenko

Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm

Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A‐chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome‐inactivating proteins. Anti‐MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin.

Mistletoe lectin A-chain unfolds during the intracellular transport

Protein conformation during intracellular routing and translocation of the ribosome‐inactivating proteins was investigated on hybridomas producing monoclonal antibodies (monAbs) against mistletoe lectin (ML). Decrease in the toxin activity towards these hybridomas is accounted for by the intracellular interaction of monAbs and the toxin resulting in the interruption of enzymatic subunit translocation into the cytosol. Obtained monAbs interacted with denatured ML A‐chain (MLA) and a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. Enzyme‐linked immunosorbent assay (ELISA) shows that monAbs recognize five epitopes in denatured MLA. Treatment of MLA by 3 M of guanidine hydrochloride leads to appearance of the epitopes. Hybridoma TA7 has been shown to be insensitive to cytotoxic action of ML. TA7 monAb as we have shown recognizes epitope 101–105, FTGTT, and inhibits the liposome aggregation induced by MLA. A study of the cytotoxicity of ML and ricin for the hybridomas revealed that the unfolding of A‐chain is probably required for intracellular transport and cytotoxic activity of ML.

Cloning and expression of mistletoe lectin III B-subunit.

Aqueous extracts of mistletoe (Viscum album L.) contain toxic proteins (lectins) MLI (viscumin), MLII, and MLIII. We previously cloned the gene encoding MLIII precursor. In the present study, a gene fragment encoding the carbohydrate-binding subunit of mistletoe toxic lectin MLIII was cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII B-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Sugar-binding activity of recombinant MLIII B-subunit was determined by ELISA. Amino acid sequence analysis of the cloned MLIII compared with known mistletoe toxins and other ribosome inactivating type II proteins (ricin, abrin a, and nigrin b B-subunits) revealed essential features of the recombinant MLIIIB primary structure that could determine sugar specificity of the lectin as well as immunomodulating and anti-tumor properties of mistletoe extracts.

Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy

DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose) polymerase 1) increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer) microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP)-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.

Structure and function of histone chaperone FACT

FACT is heterodimer protein complex and histone chaperone that plays an important role in maintaining and modifying the chromatin structure during various DNA-dependent processes. FACT is involved in nucleosome assembly de novo and in the preservation and recovery of the nucleosome structure during and after transcription, replication and DNA repair. During transcript elongation, FACT reduces the height of the nucleosome barrier and supports the maintenance of nucleosomes during and after the passage of RNA polymerase II. In this process, FACT interacts with histone H2A–H2B dimer within nucleosomes, thus, facilitating uncoiling of nucleosomal DNA from the octamer of histones. It also facilitates the subsequent recovery of the canonical nucleosome structure after transcription. FACT also plays an important role in the transformation of human cells and in maintaining the viability of tumor cells.

Inhibiting the pro-tumor and transcription factor FACT: Mechanisms

Conventional antitumor therapy is often complicated by the emergence of the so-called cancer stem cells (CSCs), which are characterized by low metabolic rates and high resistance to almost all existing therapies. Many problems of clinical oncology and a poor efficacy of current treatments in particular are ascribed to CSCs. Therefore, it is important to develop new compounds capable of eliminating both rapidly proliferating tumor cells and standard treatment-resistant CSCs. Curaxins have been demonstrated to manifest various types of antitumor activity. Curaxins simultaneously affect at least three key molecular cascades involved in tumor development, including the p53, NF-κB, and HSF1 metabolic pathways. In addition, studies of some curaxins indicate that they can inhibit the transcriptional induction of the genes for matrix metalloproteinases 1 and 8 (MMP1 and MMP8); the PI3K/AKT/mTOR signaling cascades; cIAP-1 (apoptosis protein 1) inhibitor activity; topoisomerase II; and a number of oncogenes, such as c-MYC and others. In vivo experiments have shown that the CSC population increases on gemcitabine monotherapy and is reduced on treatment with curaxin CBL0137. The data support the prospective use of FACT inhibitors as new anticancer drugs with multiple effects on cell metabolism.

A New Antigenic Epitope Appears in the Catalytic Subunit of Viscumin during Intracellular Transport

The plant toxin viscumin (60 kD) consists of B- (“binding”) and A- (“active”) subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody–toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody–toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of 1H-NMR spectroscopy.

Evaluating Parp1 domains as gossypol targets

Poly ADP-ribose Polymerase 1 (PARP1) is an important enzyme that is involved in DNA repair, replication, and transcription. Prospective anticancer drug gossypol inhibits human PARP1, but the mechanism of inhibition remains unknown. It has been shown previously that gossypol interacts with purified BRCA1 C-terminus (BRCT) domain in vitro. However, it remained unclear whether gossypol inhibits PARP1 through the BRCT domain in the context of full-length protein. Here, we report that the BRCT domain within the full-length PARP1 protein is not required for the inhibition of catalytic activity of PARP1 by gossypol. Our results, obtained using a series of PARP1 mutations and H4-dependent pathway of PARP1 activation, also show that none of the zinc fingers or other DNA binding domains of PARP1 are involved in the inhibition of the PARP1 catalytic activity by gossypol. Thus, the likely candidate target(s) for gossypol action are the other domains of PARP1, or the interdomain linkers.

Molecular mechanisms of transcriptional regulation by Poly(ADP-ribose) polymerase 1

Poly-ADP-ribosylation is a covalent posttranslational modification of nuclear proteins that plays a key role in the immediate cell response to genotoxic stress. Poly(ADP-ribose) polymerases (PARPs) synthesize long and branched ADP-ribose polymers on acceptor regulatory proteins, thereby changing their activity. Poly-ADP metabolism regulates DNA repair, the cell cycle, replication, cell senescence and death, a remodeling of the chromatin structure, and gene transcription. PARP1 is one of the most common nuclear proteins and is responsible for producing ∼90% of all ADP-ribose polymers in the cell. PARP1 inhibitors are promising as antitumor agents. At the same time, current inhibitors targeting the catalytic domain of PARP1 have a number of side effects. Considering the potential benefits PARP1 inhibitors may offer for treating many diseases, it is necessary to develop new strategies of PARP1 inhibition. PARP1 has a modular structure and possesses catalytic, transcription, and DNA-binding activities. The review focuses primarily on the role PARP1 plays in regulating transcription. The structure and functional organization of PARP1 and multiple pathways of the PARP1-dependent transcriptional regulation at the levels of chromatin remodeling, DNA methylation, and transcription are considered in detail. Studying the molecular mechanisms that regulate these processes can provide a basis for a search and design of new PARP1 inhibitors.

Atomic Force Microscopy for Investigation of Ribosome‐inactivating Proteins’ Type II Tetramerization

Biology of the toxins violently depends on their carbohydrate‐binding centres’ organization. Toxin tetramerization can lead to both increasing of lectin‐binding centres’ number and changes in their structural organization. A number and three‐dimensional localization of such centres per one molecule strongly influence on toxins’ biological properties. Ricin was used to obtain the AFM images of natural dimeric RIPsII structures as far as ricinus agglutinin was used for achievement of AFM images of natural tetrameric RIPsII forms. It is well‐known that viscumin (60 kDa) has a property to form tetrameric structures dependently on ambient conditions and its concentration. Usage of the model dimer‐tetramer based on ricin‐agglutinin allowed to identify viscumin tetramers in AFM scans and to differ them from dimeric viscumin structures. Quantification analysis produced with the NT‐MDT software allowed to estimate the geometrical parameters of ricin, ricinus agglutinin and viscumin molecules.