Biology subjects

Ruksenaite, A.

Publications and source records attributed to Ruksenaite, A..

5 recordsLinked to original sources

bioRxiv2026

Csx15 is a broad specificity ring nuclease bridging CRISPR-Cas and CBASS cyclic nucleotide signaling systems

Prokaryotic antiviral type III CRISPR-Cas and CBASS systems use cyclic nucleotides as second messengers to activate diverse effector proteins. Whereas CBASS signaling typically triggers abortive infection, type III CRISPR-Cas signaling is tightly regulated through controlled signal production and degradation. Here, we define the molecular mechanism of the CRISPR-associated Csx15 protein family and demonstrate that Csx15 functions as a broad specificity ring nuclease capable of degrading cyclic nucleotide signals from both CRISPR-Cas and CBASS pathways. Structural and biochemical analyses reveal that Csx15 adopts diverse oligomeric states and undergoes ligand-induced conformational transitions that promote filament assembly required for catalytic activity, analogous to filament-forming SAVED-domain effectors. Finally, we provide evidence that Csx15 is a close relative of SAVED domains suggesting that Csx15-like proteins may have also contributed to the evolution of CBASS cyclic nucleotide signaling.

biochemistry↗

bioRxiv2026

Deoxydinucleotides activate the bacterial anti-phage defense system ApeA

Bacteria and archaea encode diverse antiviral defense systems, many of which rely on toxic effector proteins that are activated specifically upon bacteriophage infection. However, the mechanisms by which infection is recognized and coupled to effector activation remain poorly understood for most antiviral systems. Here, we focus on ApeA, a HEPN-domain antiviral protein that confers immunity through cleavage of host tRNAs within their anticodon loops. We show that ApeA proteins form large doughnut-shaped oligomers that are activated upon ligand binding in a conserved protein pocket distinct from the catalytic center. In the Ec2ApeA variant, this pocket specifically recognizes 5'-phosphorylated deoxydinucleotides that likely arise as intermediates of host genome degradation by viral nucleases, thereby enabling Ec2ApeA to achieve a broad protection profile. Together, our results reveal how small-molecule products of virus-induced host cell destruction function as signals that activate bacterial immune defenses.

molecular biology↗

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↗

bioRxiv2024

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-{sigma} factor, CalpT, and an ECF-like {sigma} factor, CalpS. Here we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

biochemistry↗

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↗