Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 µM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

Extracellular heat shock proteins and cancer: New perspectives.

Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.