Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells.

Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profiles of both bacterial and mammalian lysine acylation enzymes, including bacterial lysine acylases PatZ, YiaC, LplA, TmcA, and YjaB, as well as mammalian acyltransferases GCN5 and Tip60, leading to discovery of distinct yet functionally important substrates and acylation sites. The concept of direct capturing substrates of PTM-enzymes via residue crosslinking may extend to the other types of amino acid residues beyond lysine, which has the potential to facilitate the investigation of diverse types of PTMs and substrate-enzyme interactive proteomics.

Sandwich hydrogel to realize cartilage-mimetic structures and performances from polyvinyl alcohol, chitosan and sodium hyaluronate.

Developing artificial substitutes that mimic the structures and performances of natural cartilage is of great importance. However, it is challenging to integrate the high strength, excellent biocompatibility, low coefficient of friction, long-term wear resistance, outstanding swelling resistance, and osseointegration potential into one material. Herein, a sandwich hydrogel with cartilage-mimetic structures and performances was prepared to achieve this goal. The precursor hydrogel was obtained by freezing-thawing the mixture of poly vinyl alcohol, chitosan and deionized water three cycles, accompanied by soaking in sodium hyaluronate solution. The top of the precursor hydrogel was hydrophobically modified with lauroyl chloride and then loaded with lecithin, while the bottom was mineralized with hydroxyapatite. Due to the multiple linkages (crystalline domains, hydrogen bonds, and ionic interactions), the compressive stress was 71 MPa. Owing to the synergy of the hydrophobic modification and lecithin, the coefficient of friction was 0.01. Additionally, no wear trace was observed after 50,000 wear cycles. Remarkably, hydroxyapatite enabled the hydrogel osseointegration potential. The swelling ratio of the hydrogel was 0.06 g/g after soaking in simulated synovial fluid for 7 days. Since raw materials were non-toxic, the cell viability was 100 %. All of the above merits make it an ideal material for cartilage replacement.

Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation.

Posttranslational modifications (PTMs) of lysine residues are major regulators of gene expression, protein-protein interactions, and protein localization and degradation. Histone lysine benzoylation is a recently identified epigenetic marker associated with active transcription, which has physiological relevance distinct from histone acetylation and can be regulated by debenzoylation of sirtuin 2 (SIRT2). Herein, we provide a protocol for the incorporation of benzoyllysine and fluorinated benzoyllysine into full-length histone proteins, which further serve as benzoylated histone probes with NMR or fluorescence signal for investigating the dynamics of SIRT2-mediated debenzoylation.

Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors.

Precise dissection of DNA-protein interactions is essential for elucidating the recognition basis, dynamics and gene regulation mechanism. However, global profiling of weak and dynamic DNA-protein interactions remains a long-standing challenge. Here, we establish the light-induced lysine (K) enabled crosslinking (LIKE-XL) strategy for spatiotemporal and global profiling of DNA-protein interactions. Harnessing unique abilities to capture weak and transient DNA-protein interactions, we demonstrate that LIKE-XL enables the discovery of low-affinity transcription-factor/DNA interactions via sequence-specific DNA baits, determining the binding sites for transcription factors that have been previously unknown. More importantly, we successfully decipher the dynamics of the transcription factor subproteome in response to drug treatment in a time-resolved manner, and find downstream target transcription factors from drug perturbations, providing insight into their dynamic transcriptional networks. The LIKE-XL strategy offers a complementary method to expand the DNA-protein profiling toolbox and map accurate DNA-protein interactomes that were previously inaccessible via non-covalent strategies, for better understanding of protein function in health and disease.

Janus Hydrogel to Mimic the Structure and Property of Articular Cartilage.

Designing hydrogels with adequate strength, remarkable swelling resistance, low friction coefficient, excellent biocompatibility, and osseointegration potential is essential for replacing articular cartilage. However, it remains challenging to integrate all these properties into one material. In this work, a Janus hydrogel was prepared from polyvinyl alcohol, chitosan, and sodium hyaluronate, followed by a one-sided dipping in situ precipitation mineralization to form a layer of hybridized hydroxyapatite (HAp), wherein the two surfaces had distinct compositions and functions. Because of the negative carboxyl groups from sodium hyaluronate, the top surface possessed a friction coefficient as low as 0.024. On account of the HAp mineralized layer, the bottom side had osteogenesis potential. Owing to the synergy of physical linkages, the hydrogel displayed compressive strength as high as 78 MPa. Furthermore, it demonstrated remarkable swelling resistance with strength retention near 100% even after soaking in PBS solution at 37 °C for 7 days. The absence of toxic chemicals maintained the merits of starting polymers and resulted in excellent biocompatibility (cell viability ≈ 100%), making it an ideal substitute for articular cartilage.

A high strength, low friction, and biocompatible hydrogel from PVA, chitosan and sodium alginate for articular cartilage.

Developing articular cartilage substitutes required a combination of high compressive strength, excellent biocompatibility and low friction. Despite great success in tough hydrogels, this combination was hardly realized. Herein, a high strength, low friction, and biocompatible hydrogel was obtained by freezing-thawing polyvinyl alcohol and chitosan aqueous solutions three times, followed with soaking in sodium alginate aqueous solution. Owing to the synergy of crystalline domains, hydrogen bonds, and ionic interactions, the obtained hydrogel exhibited high strength (maximum compressive strength = 141 MPa). Because of the reversible linkages, the gel was also creep-resistant (recovery efficiency = 93%). Benefitted from the negative carboxyl groups from sodium alginate, the water lubrication layer between the gel and the opposing surface was thickened greatly, resulting in a low coefficient of friction (0.044). The biocompatible materials and green progress led to excellent cell compatibility. All these merits made it an ideal substitute for articular cartilage.

Research progress on natural antibacterial ingredients of glass ionomer cement / 口腔疾病防治

@#Glass ionomer cement (GIC) is widely used as a common filling material in dentistry but still exhibits problems with secondary caries and fractures. Thus, the antibacterial and anti-caries performance of GIC needs to be further improved. In recent years, natural antimicrobial components have become more desirable due to their good biological properties and low drug resistance. In this review, the natural antimicrobial ingredients in GIC modification are classified, reviewed and summarized according to the different sources of antimicrobial ingredients. In terms of animal origin, chitosan and casein phosphopeptide-amorphous calcium phosphate exhibit antimicrobial properties without affecting the mechanical properties of materials; propolis and bioactive enzymes have good biocompatibility; in terms of plant origin, polyphenols help improve the antimicrobial and mechanical properties of the material; arginine has a good remineralization effect; and plant essential oils have a certain ion release effect. In terms of microbial origin, antibiotics greatly improve the antibacterial properties of materials; in addition, the combined application of natural antimicrobial ingredients also exhibited excellent performance. Despite these advantages, the optimal addition concentration and biocompatibility in vivo are questions that need to be further explored before clinical applications can be achieved.

Genetically Encoded Benzoyllysines Serve as Versatile Probes for Interrogating Histone Benzoylation and Interactions in Living Cells.

Histone posttranslational modifications (PTMs) are vital epigenetic regulators in many fundamental cell signaling pathways and diverse biological processes. Histone lysine benzoylation is a recently identified epigenetic mark associated with active transcription; however, it remains to be explored. Herein, we first report the genetic encoding of benzoyllysine and fluorinated benzoyllysines into full-length histone proteins in a site-specific manner in live cells, based on our rationally designed synthetase and fine-integrated fluorine element into benzoyllysines. The incorporated unnatural amino acids integrating unique features were demonstrated as versatile probes for investigating histone benzoylation under biological environments, conferring multiplex signals such as 19F NMR spectra with chemical clarity and fluorescence signals for benzoylation. Moreover, the site specifically incorporated lysine benzoylation within native full-length histone proteins revealed distinct dynamics of debenzoylation in the presence of debenzoylase sirtuin 2 (SIRT2). Our developed strategy for genetic encoding of benzoyllysines offers a general and novel approach to gain insights into interactions of site-specific histone benzoylation modifications with interactomes and molecular mechanisms in physiological settings, which could not be accessible with fragment histone peptides. This versatile chemical tool enables a direct and new avenue to explore benzoylation, interactions, and histone epigenetics, which will provide broad utilities in chemical biology, protein science, and basic biology research.

Light-induced efficient and residue-selective bioconjugation of native proteins via indazolone formation.

Chemical modification of proteins has emerged as a powerful tool to realize enormous applications, such as development of novel biologics and functional studies of individual protein. We report a light-induced lysine-selective native protein conjugation approach via indazolone formation, conferring reliable chemoselectivity, excellent efficiency, temporal control and biocompatibility under operationally simple and mild conditions, in vitro and in living systems. This straightforward protocol demonstrates the generality and accessibility for direct and rapid functionalization of diverse native proteins, which suggests a new avenue of great importance to bioconjugation, medicinal chemistry and chemical biology.

Light-induced primary amines and o-nitrobenzyl alcohols cyclization as a versatile photoclick reaction for modular conjugation.

The advent of click chemistry has had a profound impact on many fields and fueled a need for reliable reactions to expand the click chemistry toolkit. However, developing new systems to fulfill the click chemistry criteria remains highly desirable yet challenging. Here, we report the development of light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) as a photoclick reaction via primary amines as direct click handle, to rapid and modular functionalization of diverse small molecules and native biomolecules. With intrinsic advantages of temporal control, good biocompatibility, reliable chemoselectivity, excellent efficiency, readily accessible reactants, operational simplicity and mild conditions, the PANAC photoclick is robust for direct diversification of pharmaceuticals and biorelevant molecules, lysine-specific modifications of unprotected peptides and native proteins in vitro, temporal profiling of endogenous kinases and organelle-targeted labeling in living systems. This strategy provides a versatile platform for organic synthesis, bioconjugation, medicinal chemistry, chemical biology and materials science.

Rapid and halide compatible synthesis of 2-N-substituted indazolone derivatives via photochemical cyclization in aqueous media.

Indazolone derivatives exhibit a wide range of biological and pharmaceutical properties. We report a rapid and efficient approach to provide structurally diverse 2-N-substituted indazolones via photochemical cyclization in aqueous media at room temperature. This straightforward protocol is halide compatible for the synthesis of halogenated indazolones bearing a broad scope of substrates, which suggests a new avenue of great importance to medicinal chemistry.