RSC Chemical Biology最新文献

Streptomyces albus J1074 (now S. albidoflavus J1074) is a widely used heterologous host for natural product discovery due to its capacity to express biosynthetic gene clusters (BGCs) from diverse organisms. A derivative of this strain, S. albus Del14, enhances heterologous expression by reducing background metabolite production enabling the identification of the previously hidden BGC responsible for producing mansouramycins. In this study, we demonstrate the biosynthetic crosstalk between the native mansouramycin BGC in S. albus Del14 and introduced BGCs from three different organisms results in the production of novel compounds, some featuring rare and complex chemical scaffolds. These include malevonin, which combines NRPS- and mansouramycin-derived building blocks forming a fluorene scaffold, as well as 5′-chloromansouramycin D, a halogenated derivative of mansouramycin D. Additionally, we identified mansevorone, a compound structurally similar to mansouramycin D but utilizing a different tryptophan-derived C7 precursor. This precursor likely arises from the activation of native genes in the host S. albus Del14, triggered by SARP regulators present on the introduced BGC. These findings highlight the evolutionary significance of BGC interactions and underscore their potential as a powerful tool for discovering novel natural products, providing insights that could inform innovative strategies in biosynthetic engineering and the guided evolution of new bioactive compounds.

Bispecific agents capable of simultaneously targeting two distinct cell surface receptors promise enhanced specificity and efficacy in cancer therapy. Here, we report a strategy for the rapid optimization of compact bispecific agents using nucleic acid hybridization to display peptide ligands for both the epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition factor (MET). The self-assembly process involved 20 and 21 nucleotide (nt) long DNA-peptide conjugates and 41–46 nt template strands, which precisely controlled the spatial arrangement of the EGFR-targeting peptide GE11 and the MET-binding bicyclic peptide GE137. We introduce improved synthetic methods for the challenging construction and functionalization of GE137, enabling its efficient conjugation to oligonucleotides. Systematic variation of peptide spacing revealed a striking distance-dependent affinity profile in interactions with live A549 cells, with optimal staining observed when GE11 and GE137 were separated by 21 paired and 3 unpaired DNA nucleotides. Incorporation of a cleavable cytotoxic payload (monomethyl auristatin E) into bispecific DNA–peptide constructs led to potent, HGF-dependent cytotoxicity, underscoring the requirement for targeted internalization. Conjugation to DNA effectively masked the cytotoxic payload, unless the combined activity of GE11 and GE137 induced internalization. This work establishes that DNA-directed assembly allows precise optimization of bispecific peptide agents that are much smaller than conventional constructs, offering robust targeting and conditional cytotoxicity. These findings highlight the promise of nucleic acid scaffolds for next-generation, cell-selective therapeutics.

SIRT5, one of the human sirtuins, catalyzes the removal of acyl substitutions from lysine residues in a NAD⁺-dependent manner. In addition to the deacetylase activity, SIRT5 also demonstrates strong desuccinylase, demalonylase, and deglutarylase activity. Through deacylating a broad spectrum of cellular proteins and enzymes, SIRT5 is heavily involved in the regulation of energy metabolism, reactive oxygen species (ROS) reduction, and ammonia detoxification. Accumulating evidence also suggests SIRT5 as a potential therapeutic target for the treatment of neurodegenerative diseases, metabolic disorders, and cancer. In the current study, we report the identification and characterization of a SIRT5 modulator, reduced nicotinic acid riboside (NARH). It shows differential regulation of the distinct activities of SIRT5: it activates desuccinylation, but mildly suppresses deacetylation. NARH binds to SIRT5 in the absence of NAD⁺ and demonstrates cellular target engagement and activity. The potential NARH binding site is further investigated using a suite of biochemical and computational approaches. The current study provides greatly needed mechanistic understanding of SIRT5 regulation, as well as a novel chemical scaffold for further activator development.

The specific insertion of methyl (¹³CH₃) probes into deuterated proteins enables study of the structure, dynamics, and mechanisms of symmetric complexes up to 1 MDa by solution nuclear magnetic resonance (NMR). For asymmetric or heteromeric high molecular weight complexes, subunit-specific labelling is required to simplify spectra, reduce resonance overlap, and facilitate data interpretation. However, the instability of one component may prevent the reconstitution of a functional complex with a single labelled subunit. Here, we employed a simple, multi-step expression protocol that enabled the sequential in vitro synthesis of various subunits, each with a different isotopic labelling scheme. We exploited the open nature of the cell-free synthesis expression system by introducing a stable subunit directly into the synthesis of the unstable or poorly soluble component of the complex. This protocol was used to produce the ClpXP 26-subunit complex, in which the methyl groups were labelled on either ClpP or ClpX. The stabilisation of the newly synthesised ClpX subunits by the ClpP core enabled production of a stable and functional 760 kDa AAA+ proteolytic machine. ¹³CH₃-labelling of the alanine and methionine in ClpXP allowed acquisition of high quality 2D solution NMR spectra, the characterization of the oligomeric size using diffusion-ordered NMR spectroscopy, and observation of structural rearrangements induced by nucleotide binding.

The late-stage peptide functionalization with salicylaldehyde (SA) tags is described here as a versatile design of potential Lys-engaging, reversible-covalent ligands. This approach was applied to a known binder for NEMO, a protein involved in the activation of the pro-inflammatory transcription factor NFκB. Fluorescence anisotropy screening led to the identification of SA-tagged peptides with higher affinity than the wild-type sequence.

Adenosine diphosphate ribosylation is a significant post-translational modification implicated in various cellular processes and diseases, yet identifying its mono-ADP-ribose readers has posed considerable challenges. Previous proteomic screenings have predominantly focused on poly-ADP-ribose, resulting in the oversight of mono-ADP-ribose readers due to undefined ADP-ribose structures with randomly placed photo-crosslinking moieties. This study introduces novel, well-defined mono-ADP-ribose photoaffinity-based probes featuring distinct diazirine and benzophenone photo-crosslinkers aimed at selectively identifying mono-ADP-ribose readers. Using human HeLa protein extracts, these probes were employed in an interactomics screening, successfully uncovering numerous known and putative mono-ADP-ribose readers, including MACROD1. This study highlights the potential of these novel probes as powerful tools for exploring the mono-ADP-ribose interactome, thereby enhancing the understanding of ADP-ribosylation signaling within cellular contexts. Proteomics data are available via ProteomeXchange with identifier PXD065574.

Single-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE – a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded E. coli. Our findings establish a robust in vivo protein-labelling strategy, expanding the capabilities of SMT as a method to study the dynamics of proteins in living cells. Moreover, we observe that the strain-promoted azide–alkyne click-chemistry reaction occurs as fast as 30 min in live E. coli cells and can be used as a robust labelling reaction.

Abnormal glycosylation has been long considered a hallmark of cancer progression. Carbohydrate-binding proteins, also known as lectins, offer a unique way to target glycosylation changes in malignant cells. The present study repurposes SadP, a monomeric lectin from Streptococcus suis, to target globotriaosylceramide (Gb3), a glycosphingolipid overly abundant in many cancer types including Burkitt's lymphoma. The lectibody was designed as a fusion protein by linking the SadP to the scFv UCHT1 anti-CD3 antibody resulting in a bispecific T cell engager (BiTE)-like protein referred to as lectibody. Protein expression was carried out in Escherichia coli and the resulting lectibody was purified using affinity and size exclusion chromatography. The lectibody was tested for its specificity in binding Gb3-positive cancer cells by flow cytometry. T-cell-mediated cytotoxicity was measured in a bioluminescence-based cytotoxicity assay, and T-cell activation was assessed by evaluating CD69 and CD71 expression on PBMCs, incubated with target cells and the lectibody. The present study demonstrates that the monomeric and monovalent SadP-scFv UCHT1 lectibody can redirect T cell cytotoxicity towards Gb3⁺ Burkitt's lymphoma cells, resulting in a dose-dependent target cell lysis up to 65% in vitro at a concentration of 10 nM. In the same experimental setting, negative control cells characterized by a low or absent Gb3 content remained unaffected. Lectibody-induced T cell activation resulted in a significant increase in CD69 and CD71 surface expression in PBMCs incubated with SadP-scFv UCHT1 and Gb3 positive cancer cells. This study highlights the potential of lectins in immunotherapy for the treatment and eradication of malignant cells. The SadP-based lectibody demonstrates improved efficacy and yield when compared to the previously engineered StxB-scFv UCHT1 lectibody, therefore opening the possibility for its use in an in vivo model.

Next-generation therapies are advancing beyond small molecules and proteins toward engineered living microorganisms that interact symbiotically with their host and respond to signals precisely when and where needed. Despite progress in the field, engineering cells to both produce biopharmaceuticals and achieve site-specific recruitment remains a challenge. In this work, we genetically engineered the mating pathway of S. pombe to create a "bioprocessor" that responds to a chemical trigger, an artificial replica of the sexual pheromone of the yeast cells, the P-factor, enabling functional control over the production of Albulin as a proof-of-concept biopharmaceutical. This activation simultaneously induces the expression of hydrophobic agglutinins on the cell surface, modifying surface chemistry and adhesion properties. Exploiting this modification, we could simultaneously implement spatial control, allowing selective adhesion to a hydrophobic target surface. Adhesion control tests confirmed the fundamental role of hydrophobic interactions in this adhesion process, enabling selective cell adherence only after activation with P-factor and expression of the agglutinins, even in presence of potentially interfering cells. This approach represents an important milestone in the development of a straightforward chemically-activated multi-control mechanisms, which enable precise and programmable responses in engineered cells. Such advancements pave the way for a new generation of bio-responsive materials and therapeutic devices, including functional implants and targeted delivery systems, where engineered cells can operate in synergy with host tissues, responding to specific environmental cues to produce therapeutic agents exactly when and where they are needed.

D-Luciferin (D-LH₂) is the most used substrate for beetle luciferases in various bioluminescence applications. Here, we successfully synthesized six D-LH₂ analogues including 5′,7′-dimethoxy-D-LH₂ and 7′-methylnaphthol-D-LH₂ as novel compounds. We also developed a continuous one-pot green synthesis method to improve yields of luciferins from condensation of quinone and D-Cys (63-fold greater than the previous report). The novel D-LH₂ analogues were tested with five luciferases (Fluc, SLR, Eluc, Pmluc-WT, and Pmluc-N230S), and all the compounds emitted bioluminescence at wavelengths longer than that of D-LH₂ (>80 nm). The reaction of SLR with 5′,7′-dimethoxy-D-LH₂ gave the longest red-shifted bioluminescence at 663 nm. Remarkably, the reactions of 5′-methyl-D-LH₂ emit longer wavelengths and brighter light than those of D-LH₂ in all tested luciferases, except for Eluc. Interestingly, the novel red-shifted 5′,7′-dimethyl-D-LH₂ also provided prolonged bioluminescence with a rate of light decay slower than that of D-LH₂. We further demonstrated applications of 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂ in mammalian cell lines expressing Fluc, SLR, and Pmluc-N230S. 5′-Methyl-D-LH₂ provided about 11.2-fold greater sensitivity to detect Fluc in the HEK293T crude lysate than D-LH₂, achieving the detection with a lower number of cell lines. The red-shifted 5′,7′-dimethyl-D-LH₂ also exhibits high sensitivity when using a red light filter to monitor live cell bioluminescence. These D-LH₂ analogues, 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂, are promising substrates for future cell-based assays and real-time monitoring applications.