Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.

Delivery of Lipid Nanoparticles with ROS Probes for Improved Visualization of Hepatocellular Carcinoma.

Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.

Ribosomal leaky scanning through a translated uORF requires eIF4G2.

eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5' UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5' UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

Upregulation of Mcl-1S Causes Cell-Cycle Perturbations and DNA Damage Accumulation.

As an important regulator of apoptosis, Mcl-1 protein, a member of the Bcl-2 family, represents an attractive target for cancer treatment. The recent development of novel small molecule compounds has allowed Mcl-1-inhibitory therapy to proceed to clinical trials in cancer treatment. However, the possible adverse effects of either direct inhibition of Mcl-1 or upregulation of Mcl-1S, proapoptotic isoform resulting from alternative splicing of Mcl-1, remain unclear. Here, we investigated changes in Mcl-1S levels during cell cycle and the cell cycle-related functions of Mcl-1 isoforms to address the above-mentioned concerns. It was shown that an anti-mitotic agent monastrol caused accumulation of Mcl-1S mRNA, although without increasing the protein level. In contrast, both mRNA and protein levels of Mcl-1S accrued during the premitotic stages of the normal cell cycle progression. Importantly, Mcl-1S was observed in the nuclear compartment and an overexpression of Mcl-1S, as well as knockdown of Mcl-1, accelerated the progression of cells into mitosis and resulted in DNA damage accumulation. Surprisingly, a small molecule inhibitor of Mcl-1, BH3-mimetic S63845, did not affect the cell cycle progression or the amount of DNA damage. In general, upregulated Mcl-1S protein or genetically inhibited Mcl-1L were associated with the cell cycle perturbations and DNA damage accumulation in normal and cancer cells. At the same time, BH3-mimetic to Mcl-1 did not affect the cell cycle progression, suggesting that direct inhibition of Mcl-1 is devoid of cell-cycle related undesired effects.

Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA.

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5'- and 3'-end and studied their photo physical properties. We demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples).

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.