Aggregation of antimicrobial peptides (AMPs) enhances their efficacy by destabilising the bacterial cell wall, membrane, and cytosolic proteins. Developing aggregation-prone AMPs offers a promising strategy to combat antibiotic resistance, though predicting such AMPs and understanding bacterial responses remain challenging. Octopus bimaculoides, a cephalopod species, lacks known AMP gene families, yet its protein fragments were used to predict AMPs via artificial intelligence tools. Four peptides (Oct-P1, Oct-P2, Oct-P3, and Oct-P4) were identified based on their aggregation propensity. Among them, Oct-P2 reduced the viability of Escherichia coli and Staphylococcus aureus by up to 90%, confirmed by confocal laser scanning microscopy and scanning electron microscopy. It further aggregated plasmid DNA in vitro, and the presence of extracellular DNA reduced their antibacterial activity. With knockout mutants, it revealed that Oct-P2 was internalised into bacterial cells, possibly through membrane transport proteins, enhancing its antibacterial effect. Aggregation-induced emission assays and molecular dynamics simulations revealed that Oct-P2 aggregates with transcription promoter DNA, inhibiting transcription and translation in vitro. This dual-target mechanism not only highlights the potential of Oct-P2 as a lead template for new antimicrobial drug development, but also opens a new window for discovering AMPs from protein fragments against the upcoming challenge of bacterial infections.

α-Cyanostilbene represents one of the easily functionalized aggregation-induced emission (AIE) scaffolds. It has been widely adopted for the construction of fluorescent materials for broad applications. Here, we further expanded the utilization of α-cyanostilbene derivatives for the detection of hypoxia or proteostasis imbalance in live cells. Four different amine containing donors were introduced to construct α-cyanostilbene derivatives (R-ASC) with donor-acceptor scaffolds. Equipped with the cysteine (Cys) reactive group, maleimide (MI), R-ASC-MI shows fluorescence turn-on property upon binding with unfolded proteins in vitro and in live cells under proteostatic stress. By virtue of R-ASC-MI, the level of unfolded protein loads in cells can be quantified by flow cytometry, or visualized under microscope. Furthermore, we also characterized the performance of R-ASC-NO2, synthetic precursors of R-ASC-MI, in cellular hypoxia. R-ASC-NO2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN3) induced or atmospheric (1 % O2) hypoxia. Together, the advantages of easy modification and high signal-to-noise ratio of new α-cyanostilbene derivatives reported in this work highlight the great potential of α-cyanostilbene in constructing functional biosensors and many other domains.α-Cyanostilbene represents one of the easily functionalized aggregation-induced emission (AIE) scaffolds. It has been widely adopted for the construction of fluorescent materials for broad applications. Here, we further expanded the utilization of α-cyanostilbene derivatives for the detection of hypoxia or proteostasis imbalance in live cells. Four different amine containing donors were introduced to construct α-cyanostilbene derivatives (R-ASC) with donor-acceptor scaffolds. Equipped with the cysteine (Cys) reactive group, maleimide (MI), R-ASC-MI shows fluorescence turn-on property upon binding with unfolded proteins in vitro and in live cells under proteostatic stress. By virtue of R-ASC-MI, the level of unfolded protein loads in cells can be quantified by flow cytometry, or visualized under microscope. Furthermore, we also characterized the performance of R-ASC-NO2, synthetic precursors of R-ASC-MI, in cellular hypoxia. R-ASC-NO2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN3) induced or atmospheric (1 % O2) hypoxia. Together, the advantages of easy modification and high signal-to-noise ratio of new α-cyanostilbene derivatives reported in this work highlight the great potential of α-cyanostilbene in constructing functional biosensors and many other domains.

Site-selective chemical modifications of proteins have emerged as a potent technology in chemical biology, materials science, and medicine, facilitating precise manipulation of proteins with tailored functionalities for basic biology research and developing innovative therapeutics. Compared to traditional recombinant expression methods, one of the prominent advantages of chemical protein modification lies in its capacity to decorate proteins with a wide range of functional moieties, including non-genetically encoded ones, enabling the generation of novel protein conjugates with enhanced or previously unexplored properties. Among these, approaches for dual or multiple protein modifications are increasingly garnering attention, as it has been found that single modifications of proteins are inadequate to meet current demands. Therefore, in light of the rapid developments in this field, this review provides a timely and comprehensive overview of the latest advancements in chemical and biological approaches for protein dual functionalization. It further discusses their advantages, limitations, and potential future directions in this relatively nascent area.

The efficiency of traditional antibiotics has been undermined by the proliferation of antibiotic-resistant pathogenic microorganisms, necessitating the pursuit of innovative therapeutic agents. Antimicrobial peptides (AMPs), which are part of host defence peptides found ubiquitously in nature, exhibiting a wide range of activity towards bacteria, fungi, and viruses, offer a highly promising candidate solution. The efficacy of AMPs can frequently be augmented via alterations to their amino acid sequences or structural adjustments. Given the vast reservoir of marine life forms and their distinctive ecosystems, marine AMPs stand as a burgeoning focal point in the quest for alternative peptide templates extracted from natural sources. Advances in identification and characterization techniques have accelerated the discoveries of marine AMPs, thereby stimulating AMP customization, optimization, and synthesis research endeavours. This review presents an overview of recent discoveries related to the intriguing qualities of marine AMPs. Emphasis will be placed upon post-translational modifications (PTMs) of marine AMPs and how they may impact functionality and potency. Additionally, this review considers ways in which marine PTM might support larger-scale, heterologous AMP manufacturing initiatives, providing insights into translational applications of these important biomolecules.