Biology subjects

Ivanov, A. V.

Publications and source records attributed to Ivanov, A. V..

6 recordsLinked to original sources

bioRxiv2025

Observing intersubunit dynamics in single yeast ribosomes

Translation is accompanied by the rotation of the small and large ribosomal subunits relative to each other. Here, we use single-molecule Forster resonance energy transfer between fluorophores introduced into ribosomal proteins uS15 and eL30 to follow the intersubunit dynamics of Saccharomyces cerevisiae ribosomes. Similar to their bacterial counterparts, yeast ribosomes are observed to sample two predominant FRET states corresponding to the nonrotated (NR) and rotated (R) conformations. Our data yield further evidence that intersubunit rotation is coupled to tRNA transitions between the classical and hybrid binding states. In particular, the transition from the NR to R conformation is inhibited by the antibiotic cycloheximide, which is known to bind to the E site of the large subunit and prevent the movement of deacylated tRNA into the hybrid P/E state. The elongation cycle, which comprises tRNA binding, peptide transfer, and mRNA-tRNA translocation, is accompanied by switching from NR to R, and then back to the NR conformation. We find that fungal elongation factor 3 (eEF3) stabilizes the NR conformation of the ribosome. Our data are consistent with the model suggesting that eEF3 facilitates E-site tRNA release at the late step of mRNA-tRNA translocation, following the reverse intersubunit rotation induced by the universally conserved elongation factor 2 (eEF2). Our uS15-eL30 smFRET assay provides the basis for investigating eukaryotic mechanisms of translation regulation, including ribosome pausing, stalling, and frameshifting.

biophysics↗

bioRxiv2025

A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 minutes. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

biochemistry↗

bioRxiv2024

Eukaryotic initiation factors eIF4F and eIF4B promote translation termination upon closed-loop formation

Eukaryotic translation initiation factor eIF4F, comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with PABP bound to the poly(A) tail of mRNA, thereby forming a closed-loop structure. PABP, in turn, interacts with eRF3, stimulating translation termination. Here, we employed a reconstituted mammalian system to directly demonstrate that eIF4F potently enhances translation termination. Specifically, eIF4A and eIF4B promote the loading of eRF1 into the A site of the ribosome, while eIF4G1 stimulates the GTPase activity of eRF3 and facilitates the dissociation of release factors following peptide release. We also identified MIF4G as the minimal domain required for this activity and show that eIF4G2/DAP5 can also promote termination. Our findings provide compelling evidence that the closed-loop mRNA structure facilitates translation termination, with PABP and eIF4F directly involved in this process.

molecular biology↗

bioRxiv2023

Grubraw, a chemogenetic generator of mitochondrial pyruvate, reveals new mechanisms of mitochondrial metabolic control that underlie the Warburg effect

Upregulation of glycolysis and downregulation of mitochondrial oxidative phosphorylation, termed as the Warburg effect, are characteristic of tumor cells1,2. Restriction of pyruvate flux into the mitochondrial matrix is one of the major mechanisms underlying this phenomenon3. Warburg-type metabolism is beneficial for rapidly proliferating cells, however its function remains unclear. Moreover, it is unknown what the metabolic consequences of activation of mitochondrial respiration in Warburg-type cancer cells are. Here we created a chemogenetic instrument, Grubraw, that generates pyruvate directly in the mitochondrial matrix bypassing restricted pyruvate influx. In cancer cells, Grubraw-driven pyruvate synthesis in the matrix increased mitochondrial membrane potential, oxygen consumption rate, and the amounts of TCA cycle intermediates. In a mouse model of human melanoma xenografts, chemogenetic activation of mitochondria caused a decrease in tumor growth rate. Surprisingly, cancer cells actively exported pyruvate generated by Grubraw in the mitochondria into the extracellular medium. In addition, activation of mitochondria induced downregulation of transcription of the genes that drive cell cycle progression, cell proliferation and DNA replication. Our results demonstrate that cells with Warburg-type metabolism use previously unknown mechanisms of carbon flux control to dispose of excessive mitochondrial pyruvate, and activation of mitochondria in these cells downregulates cellular proliferation.

cancer biology↗

bioRxiv2022

SARS-CoV-2 Subunit Virus-Like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study

Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of "Betuvax-CoV-2", a vaccine developed as a subunit vaccine containing a recombinant RBD-Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant ("Betuspheres"). The aim of the study was to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, "Betuvax-CoV-2" was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway.

pharmacology and toxicology↗

bioRxiv2020

Virion-associated spermidine transmits with Rift Valley fever virus particles to maintain infectivity

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules is diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively-charged molecules involved in cell cycle, translation, and nucleic acid synthesis, among other cellular functions. Polyamines also support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative-sense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity. We show that RVFV replication is facilitated by any of the three biogenic polyamines; however, we specifically find spermidine associated with purified virions. Using a panel of polyamine homologs, we observe that virions can also associate with (R)-3-methylspermidine and norspermidine, though not with other less homologous molecules. Using polyamine reporter cells, we demonstrate that virion-associated polyamines transmit from one infected cell to another. Finally, we find that virions devoid of polyamines are unstable and cannot be supplemented with exogenous polyamines to regain stability or infectivity. These data highlight a unique role for polyamines, and spermidine in particular, in maintaining virus infectivity, a function not previously appreciated. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.

microbiology↗