RSC Chemical Biology最新文献

Genetically encoded Ras biosensors have been instrumental in illuminating the spatiotemporal dynamics of Ras activity since the beginning of the imaging revolution of the early 21st century. In general, these sensors employ Ras sensing units coupled with fluorescent proteins. These biosensors have not only helped elucidate Ras signalling dynamics at the plasma membrane but also revealed novel roles for Ras signalling within subcellular compartments such as the Golgi apparatus. In this review, we discuss the different classes of biosensors used to measure Ras activity and discuss their importance in uncovering new roles for Ras activity in cellular signalling and behavior.

Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein–protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we’ve combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible via E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.

The occurrence of non-canonical nucleoside structures in RNA of biological or synthetic origin has encountered several recent boosts in attention, namely in the context of RNA modifications, and with an eye to RNA vaccines. New nucleoside structures introduce added functionality and function into biopolymers that are otherwise rather homogenous in their chemical structure. Here, we report the discovery of a presumed RNA modification that was identified by combination of liquid chromatography–tandem mass spectrometry (LC–MS/MS) with stable isotope labelling as a dimer of the known RNA modification 4-thiouridine (s⁴U). The disulfide-linked structure, which had previously been synthetically introduced into RNA, was here formed spontaneously in isolates of E. coli tRNA. Judicious application of stable isotope labelling suggested that this presumed new RNA modification was rather generated ex vivo by oxidation with ambient oxygen. These findings do not only underscore the need for caution in the discovery of new RNA modifications with respect to artifacts, but also raise awareness of an RNA vulnerability, especially to oxidative damage, during its transport or storage.

Passive membrane permeability is an important property in drug discovery and biological probe design. To elucidate the cell-penetrating ability of oxadiazole-containing (Odz) peptides, we employed the Chloroalkane Penetration Assay. The present study demonstrates that Odz cyclic peptides can be highly cell-penetrant depending on the position of specific side chains and the chloroalkane tag. Solution NMR shows that Odz cyclic peptides adopt a β-turn conformation. However, despite observing high cell penetration, we observed low passive permeability in experiments with artificial membranes. These findings highlight the complexity of controlling cell penetration for conformationally sensitive macrocycles and suggest that Odz cyclic peptides may provide a framework for designing cell-penetrant cyclic peptides.

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We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

Proteolysis-targeting chimeras or PROTACs are hetero-bifunctional molecules designed to mediate the disposal of a target protein via recruitment of the ubiquitination–proteasome degradation machinery. Because of the chimeric nature of such molecules, their synthesis requires a key step of “assembling” whether in the lab or in situ. Furthermore, targeted PROTACs often are hetero-trifunctional and require a second “assembling” step. Click chemistry has the unique advantages of tethering two or more molecular entities of choice under near physiological conditions and therefore has been applied to the development of PROTACs in various ways. This review provides a succinct summary of this field with a critical analysis of various factors that need to be considered for optimal results. Specifically, we examine issues including applications of click chemistry in in situ assembly for improved delivery, conjugation with a targeting group for selectivity, rapid synthesis for linker optimization, and lysosomal degradation of extracellular and membrane-associated proteins. We also examine reaction kinetics issues whenever possible or warranted.

In addition to their classical role in ATP generation, mitochondria also contribute to Ca²⁺ buffering, free radical production, and initiation of programmed cell death. Mitochondrial dysfunction has been linked to several leading causes of morbidity and mortality worldwide including neurodegenerative, metabolic, and cardiovascular diseases as well as several cancer subtypes. Thus, there is growing interest in developing drug-delivery vehicles capable of shuttling therapeutics directly to the mitochondria. Here, we functionalized the conventional 10,12-pentacosadiynoic acid/1,2-dimyristoyl-sn-glycero-3-phosphocholine (PCDA/DMPC)-based liposome with a mitochondria-targeting triphenylphosphonium (TPP) cationic group. A fluorescent dansyl dye (DAN) group was also included for tracking mitochondrial drug uptake. The resultant PCDA-TPP and PCDA-DAN conjugates were incorporated into a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based lipid bilayer, and these modified liposomes (Lip-DT) were studied for their cellular toxicity, mitochondrial targeting ability, and efficacy in delivering the drug Doxorubicin (Dox) to human colorectal carcinoma (HCT116) and human breast (MCF7) cancer cells in vitro. This Lip-DT-Dox exhibited the ability to shuttle the encapsulated drug to the mitochondria of cancer cells and triggered oxidative stress, mitochondrial dysfunction, and apoptosis. The ability of Lip-DT-Dox to trigger cellular toxicity in both HCT116 and MCF7 cancer cells was comparable to the known cell-killing actions of the unencapsulated drug (Dox). The findings in this study reveal a promising approach where conventional liposome-based drug delivery systems can be rendered mitochondria-specific by incorporating well-known mitochondriotropic moieties onto the surface of the liposome.

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure–activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.