Biology subjects

Manakova, E.

Publications and source records attributed to Manakova, E..

5 recordsLinked to original sources

bioRxiv2025

Activation of SPARDA defense system by filament assembly reveals beta-relay signaling mechanism widespread in prokaryotic Argonautes

Present in all three domains of life, Argonaute (Ago) proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets and are involved in RNA silencing (eukaryotes) or host defense against invading DNA (prokaryotes). Here, we show that the SPARDA (short prokaryotic Argonaute, DNase associated) system functions in anti-plasmid defense. Upon detection of invading plasmid DNA, it degrades both the invader and host DNA, inducing cell death and preventing further spread of the invader. Upon activation, the recognition signal of the bound guide/target duplex is relayed to other functional SPARDA sites through a structural region, which we termed beta-relay. The associated dramatic conformational changes trigger the formation of a filament, in which the DREN nuclease domains form tetramers poised to cleave dsDNA. Furthermore, we identified the presence of beta-relay in all pAgo clades, providing new insights into the structural mechanisms of pAgo proteins.

biochemistry↗

bioRxiv2024

Targeted anticancer pre-vinylsulfone covalent inhibitors of carbonic anhydrase IX

We designed novel pre-drug compounds that transform into an active form that covalently modifies particular His residue in the active site, a difficult task to achieve, and applied to carbonic anhydrase (CAIX), a transmembrane protein, highly overexpressed in hypoxic solid tumors, important for cancer cell survival and proliferation because it acidifies tumor microenvironment helping invasion and metastases processes. The designed compounds have several functionalities: 1) primary sulfonamide group recognizing carbonic anhydrases (CA), 2) high-affinity moieties specifically recognizing CAIX among all CA isozymes, and 3) forming a covalent bond with the His64 residue. Such targeted covalent compounds possess both high initial affinity and selectivity for the disease target protein followed by complete irreversible inactivation of the protein via covalent modification. Our designed prodrug candidates bearing moderately active pre-vinyl sulfone esters or weakly active carbamates optimized for mild covalent modification activity to avoid toxic non-specific modifications and selectively target CAIX. The lead inhibitors reached 2 pM affinity, highest among known CAIX inhibitors. The strategy could be used for any disease drug target protein bearing a His residue in the vicinity of the active site.

biophysics↗

bioRxiv2021

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2-protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

molecular biology↗

bioRxiv2020

Oriented Soft DNA Curtains for Single Molecule Imaging

Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

biophysics↗

bioRxiv2020

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

BackgroundArgonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. ResultsIn this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal {beta}-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the dimerization surface mutant AfAgo{Delta}, forms a homodimer both in the apo-form and when bound to a nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds two ends of a linear DNA fragment, forming a relatively stable DNA loop. ConclusionOur results show that contrary to other characterized Ago proteins, AfAgo is a stable homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

biochemistry↗