Abnormal intracellular phase transitions in mutant hnRNP A1 may underlie the development of several neurodegenerative diseases. The risk of these diseases increases upon C9Orf72 repeat expansion and the accumulation of the corresponding G-quadruplex (G4)-forming RNA, but the link between this RNA and the disruption of hnRNP A1 homeostasis has not been fully explored so far. Our aim was to clarify the mutual effects of hnRNP A1 and C9Orf72 G4 in vitro. Using various optical methods and atomic force microscopy, we investigated the influence of the G4 on the formation of cross-beta fibrils by the mutant prion-like domain (PLD) of hnRNP A1 and on the co-separation of the non-mutant protein with a typical SR-rich fragment of a splicing factor (SRSF), which normally drives the assembly of nuclear speckles. The G4 was shown to act in a holdase-like manner, i.e., to restrict the fibrillation of the hnRNP A1 PLD, presumably through interactions with the PLD-flanking RGG motif. These interactions resulted in partial unwinding of the G4, suggesting a helicase-like activity of hnRNP A1 RGG. At the same time, the G4 was shown to disrupt hnRNP A1 co-separation with SRSF, suggesting its possible contribution to pathology through interference with splicing regulation.

Pharmacological chaperones of heterogeneous nuclear ribonucleoproteins (hnRNPs) show promise as potential neuroprotective drug candidates. They are expected to prevent the accumulation of neurotoxic hnRNP biocondensates and aggregates, which are hallmarks of severe degenerative diseases. Here, we present the first rational design of oligonucleotide chaperones of hnRNP A1. This design was inspired by previous studies on the specificity of the RNA recognition motif (RRM) and the RGG motif of hnRNP A1 for endogenous nucleic acids. To obtain robust and specific chaperones, we combined an RRM-binding sequence with an RGG-binding G-quadruplex oligonucleotide that inhibits hnRNP A1 aggregation and introduced various modifications into the sugar-phosphate backbone of the oligonucleotide. Modifications that locked the RRM-binding sequence in a conformational state characteristic of RNA improved chaperone affinity and activity. The former was assessed using microscale thermophoresis assays, while the latter was evaluated using fluorimetry and microscopy. The leading chaperone bound to hnRNP A1 at micromolar concentrations and inhibited the assembly of its condensates and amyloid-like aggregates (fibrils) by over 90%.

The growing threat of infectious diseases requires novel therapeutics with different mechanisms of action. Antimicrobial peptides (AMPs), which are crucial for innate immunity, are a promising research area. The medicinal leech (Hirudo medicinalis) is a potential source of bioactive AMPs that are vital while interacting with microorganisms. This study aims to investigate the antimicrobial properties of peptides found in the H. medicinalis genome using a novel high-throughput screening method based on the expression of recombinant AMP genes in Escherichia coli. This approach enables the direct detection of AMP activity within cells, skipping the synthesis and purification steps, while allowing the simultaneous analysis of multiple peptides. The application of this method to the first identified candidate AMPs from H. medicinalis resulted in the discovery of three novel peptides: LBrHM1, NrlHM1 and NrlHM2. These peptides, which belong to the lumbricin and macin families, exhibit significant activity against E. coli. Two fragments of the new LBrHM1 homologue were synthesised and studied: a unique N-terminal fragment (residues 1–23) and a fragment (residues 27–55) coinciding with the active site of lumbricin I. Both fragments exhibited antimicrobial activity in a liquid medium against Bacillus subtilis. Notably, the N-terminal fragment lacks homologues among previously described AMPs.

Background/Objectives: The global rise in antibiotic resistance necessitates the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), key components of innate immunity, are promising candidates. This study aimed to develop novel therapeutic peptides with enhanced properties through the mutagenesis of natural AMPs and high-throughput screening. Methods: We constructed mutant libraries of three broad-spectrum AMPs—melittin, cecropin, and Hm-AMP2—using mutagenesis with partially degenerate oligonucleotides. Libraries were expressed in Escherichia coli, and antimicrobial activity was assessed through bacterial growth kinetics and droplet serial dilution assays. Candidate molecules were identified by DNA sequencing, and the most promising variants were chemically synthesized. Antimicrobial activity was determined by minimal inhibitory concentration (MIC) against E. coli and Bacillus subtilis, while cytotoxicity was evaluated in human Expi293F cells (IC₉₀) viability. The therapeutic index was calculated as the ratio of an AMP’s cytotoxic concentration to its effective antimicrobial concentration. Results: Mutant forms of melittin (MR1P7, MR1P8) showed significantly reduced cytotoxicity while retaining antimicrobial activity. Cecropin mutants exhibited reduced efficacy against E. coli, but variants CR2P2, CR2P7, and CR2P8 gained activity against Gram-positive bacteria. Mutagenesis of Hm-AMP2 generally decreased activity against E. coli, though two variants (A2R1P5 and A2R3P6) showed retained or enhanced efficacy against B. subtilis while maintaining low cytotoxicity. Conclusions: The proposed strategy successfully generated peptides with improved therapeutic profiles, including reduced toxicity or a broader spectrum of antimicrobial activity, despite not improving all parameters. This approach enables the discovery of novel bioactive peptides to combat antibiotic-resistant pathogens.