RSC Chemical Biology最新文献

Phage display is a powerful platform for ligand evolution, but conventional phage display libraries are confined to the twenty canonical amino acids, greatly limiting the chemical space that these libraries can be used to explore. Here we present an approach to expand the molecular diversity of phage-displayed peptides that combines unnatural amino acid mutagenesis with chemical post-translational modification. By incorporating azide-functionalized unnatural amino acids into phage-displayed peptides and applying optimized conditions for copper-catalysed azide–alkyne cycloaddition, we achieve quantitative and selective peptide modification with a series of alkyne-functionalized small molecules. This approach provides a general platform for constructing chemically augmented phage-displayed libraries with broad utility in ligand discovery.

Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

Near-infrared photoimmunotherapy (NIR-PIT) employing an antibody labeled with a silicon phthalocyanine dye, IR700, was approved as a minimally invasive treatment for unresectable recurrent head and neck cancer in Japan in 2020. However, further derivatization of IR700 is needed to increase the efficiency of cancer treatment. Here, we developed SiPc-1 as an IR700 analog, in which the linker was constructed using click chemistry to simplify the synthetic scheme and its position was switched from α to β on the benzene ring of phthalocyanine to eliminate intramolecular steric repulsion. We evaluated the cleavage rate of the water-soluble axial moieties of SiPc-1 upon photoirradiation, the cytotoxicity, and the morphological change (blebbing) of treated cells upon photoirradiation. We performed gene expression and protein expression analyses to find a target antigen selectively expressed on cells infected with human T-cell lymphotropic virus type 1 (HTLV-1), the causative virus of adult T-cell leukemia/lymphoma (ATL), and identified CD25 as a suitable target antigen. An anti-CD25 antibody, basiliximab, labeled with SiPc-1 (bas-SiPc-1) showed selective toxicity towards HTLV-1-infected cultured cells and ATL patients’ peripheral blood mononuclear cells upon photoirradiation.

Protein lipidation, particularly palmitoylation (attachment of a 16-carbon fatty acid), regulates cellular behaviors by controlling protein function at lipid membranes. In this study, we prepared a series of lipidated green fluorescent proteins (“EGFP–lipids”) with various alkyl chain lengths (C8 to C22). Using model lipid membranes and Jurkat (human T lymphocyte) cells, we evaluated how lipidation affects the membrane interactions and vesicular transport from the membrane of these protein–lipid constructs. Our findings demonstrate that elongation of the alkyl chain profoundly affects both lateral membrane diffusion and vesicular transport of the EGFP–lipids. Only artificially lipidated proteins that mimic in vivo lipidation exhibited cellular dynamics in response to external signals, which highlights the significance of the natural selection of palmitic acid to maximize the function of proteins on lipid membranes. This insight can also be useful in membrane engineering using artificial protein lipidation techniques, potentially accelerating medical and industrial developments.

Cell-penetrating peptides (CPPs) can internalize ubiquitously in cells. To explore the specific targeting issue of CPPs, we used glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs). The Otx2 sequence preferentially recognizes highly sulfated chondroitin (CS) and the En2 one, heparan sulfates (HS) GAGs. The two HPs internalize in specific cells thanks to their GAG-targeting sequence. We studied the capacity of chimeric peptides containing a GAG-targeting and a penetratin-like sequences to enter into various cell lines known to express different levels and types of GAGs. Since GAGs are found at the vicinity the membrane lipid bilayer, we also analyzed the putative binary and ternary interactions between heparin (HI), (4S,6S)-CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those chimeric peptides. Altogether, our results demonstrate the existence of Ca²⁺-dependent interactions between GAGs and PC lipid bilayers, the major phospholipid headgroup found in animal cell plasma membrane. In addition, the interaction of CS-E (but not HI), with PC favors the binding of the chimeric CS-E-recognition motif-penetratin-like peptide and its subsequent crossing of the lipid membrane to access directly to the cytosol of cells. Altogether, this study brings further understanding of translocation mechanism of CPPs, which requires specific GAGs at the cell-surface. It also shed light on the role of GAGs in the cell transfer specificity and paracrine activity of HPs.

A common approach in living medicine engineering is modifying cell surfaces with nanomedicines to form nanoparticle–cell conjugates. Despite various available strategies, limited research has examined how conjugation strategies affect the efficiency and stability of the delivery systems. Herein, we prepared polymeric nanoparticles (NPs) with protein payloads and modified them with different linkers. These NPs were conjugated to primary splenocytes using either covalent or electrostatic interactions, followed by flow cytometry analysis to evaluate the conjugating efficiency and stability. The results demonstrated that electrostatic interactions were more effective in achieving conjugation, whereas covalent interactions provided greater stability. Furthermore, the linker density on the nanoparticle surface also affected the stability. After three days of in vitro culture, NPs with fewer linkers were predominantly internalized by the splenocytes, whereas those with more linkers partially remained on the cell surface. Overall, this study provides fundamental insights into nanoparticle–cell conjugation, thereby contributing to living medicine design and engineering for therapeutic applications.

Metabolic oligosaccharide engineering (MOE) is a transformative technology, enabling the chemical labeling and subsequent analysis of glycans. Central to this method are monosaccharide analogs, termed metabolic chemical reporters (MCRs), that contain abiotic functional groups that can undergo an increasing number of bioorthogonal reactions. Typically, these abiotic groups were designed to be as small as possible, allowing them to be tolerated by metabolic enzymes and glycosyltransferases that transform MCRs into donor sugars and add them into glycans, respectively. This generality allows MCRs to be used by a variety of cells and tissues but can also be a limitation in their application to investigate glycosylation of specific cell-types in multicellular systems. Here, we review different methods that are beginning to transition MCRs into cell selective tools, with the potential to increase the already large impact these compounds have had on glycoscience.

Members of the Jumonji C (JmjC) subfamily of non-heme Fe(II) and 2-oxoglutarate (2OG) dependent N^ε-lysine demethylases have established roles in catalysing demethylation of N^ε-methylated lysine residues in core histones; their roles in accepting linker H1 histones as substrates have been less well explored. We report studies on the H1 substrate specificity of human JmjC lysine demethylases (KDMs), specifically KDM3A-C, KDM4A, KDM4D, KDM4E, KDM5D, and KDM6B, for mono-, di- and trimethylated N^ε-lysine residues in peptide fragments of the N-terminal tail of human linker histone H1 isoforms (H1.2, H1.3, H1.4 and H1.5). The KDM4s, but not the other tested JmjC KDMs, catalysed demethylation of tri- and dimethylated H1 peptide isoforms with activities: KDM4E > KDM4D > KDM4A. The order of substrate preference for KDM4E was H1.2K26me3 > H1.5K26me3 ≈ H1.3K24me3 > H1.2K25me3 ≈ H1.4K25me3. For KDM4D, the most efficient tested substrate was H1.5K26me3. Among the dimethylated H1 peptide isoforms, H1.3K24me2 appeared to be the most efficient KDM4E substrate, with comparable activity to the core histone H3K9me2 substrate. The results demonstrate that JmjC KDM4s can accept the N-terminal H1 tails as substrates, further highlighting the potential for flexibility in substrate and product selectivity of the JmjC KDMs, in particular, within the KDM4 subfamily. Molecular and cellular investigations on JmjC KDM-catalysed H1 demethylation are of molecular and biomedical interest.

Achieving skin regeneration following destruction of the epidermis and dermis (e.g. full-thickness wounds) has remained an unachieved goal. The wound healing response is complex and consists of multiple overlapping phases which are tightly choreographed by the ebb and flow of effector molecules. Mimicking this spatiotemporal aspect in pro-regenerative biomaterials may enhance their efficacy and eventually lead to skin regeneration. However, robust spatiotemporal signalling has remained difficult to achieve. The field of bioorthogonal click and click-to-release chemistry may be key to creating spatiotemporal signalling biomaterials. The ability to safely and effectively conjugate or release molecules in complex biological environments has transformed many areas of research. In this review, we aim to highlight the complex nature of wound healing and address how click and click-to-release chemistry approaches could contribute to the development of biomaterials with spatiotemporal control over effector molecules.

Cytochrome P450 enzymes in ribosomally synthesized and post-translationally modified peptides (RiPPs) catalyze C–C, C–N, or C–O cross-linking reactions in the biosynthesis of biaryl cyclophane natural products. Here, we manually identified 127 homologous P450s linked to putative precursor peptides containing the YPW motif. Through in vivo functional studies in Escherichia coli, the newly identified enzyme MpoB from Micromonospora polyrhachis DSM 45886 was found to catalyze the formation of a cross-link between Tyr-C3 and Trp-N1 at the YPW motif. This result provides an additional toolkit for cross-linked peptide modification.