CXC-Mediated Cellular Uptake of Miniproteins: Forsaking “Arginine Magic”

Miniproteins have a size between that of larger biologics and small molecules and presumably possess the advantages of both; they represent an expanding class of promising scaffolds for the design of affinity reagents, enzymes, and therapeutics. Conventional strategies to promote cellular uptake of miniproteins rely on extensive grafting or embedding of arginine residues. However, the requirement of using cationic arginines would cause problems to the modified miniproteins, for example, low solubility in solutions (proneness of aggregation) and potential toxicity, which are open secrets in the peptide and protein communities. In this work, we report that the cell-permeability of cationic miniproteins can be further markedly increased through appending a magic CXC (cysteine-any-cysteine) motif, which takes advantage of thiol–disulfide exchanges on the cell surface. More importantly, we discovered that the high cell permeability of the CXC-appended miniproteins can still be preserved when the embedded arginines are all substituted with lysine residues, indicating that the “arginine magic” essential to almost all cell-permeable peptides and (mini)­proteins is not required for the CXC-mediated cellular uptake. This finding provides a new avenue for designing highly cell-permeable miniproteins without compromise of potential toxicity and stability arising from arginine embedding or grafting

Niflumic Anion Intercalated Layered Double Hydroxides with Mechano-Induced and Solvent-Responsive Luminescence

Stimuli-responsive luminescent materials play an important role in fluorescent switches, optical storage devices and smart sensors. In this work, we report a mechano-induced and solvent stimuli-responsive luminescent change by the assembly of a typical aggregation-induced-emissive (AIE) molecule, niflumic acid (NFC), into the interlayer region of Zn–Al-layered double hydroxides (LDHs) with heptanesulfonate (HPS) as a cointercalation guest. The structure, chemical composition, and thermostability of the as-prepared NFC-HPS/LDHs composites were characterized by X-ray diffraction, elemental analysis, and thermogravimetry and differential thermal analysis (TG-DTA). Fluorescence spectra demonstrate that the sample with 5% NFC initial molar percentage, with respect to the interlayer guests, exhibits the optimal luminescent intensity. The NFC-HPS/LDH (5%) sample also exhibits the most obvious luminescent mechano-response with a 16 nm blue-shift and increase in the fluorescent intensity after grinding, while the pristine NFC solid shows little to no mechano-responsive behavior. Moreover, the NFC-HPS/LDH (5%) also presents reversible luminescent response to different volatile organic compounds (VOCs) (such as tetrahydrofuran, methanol, acetone, toluene, and chloroform). Therefore, this work not only gives a detailed description on the dual stimuli (mechanics and solvent)-responsive luminescence for future sensor applications but also supplies a deep understanding of the optical properties of the new AIE molecule within the confined LDH layers

Orthogonal Cysteine–Penicillamine Disulfide Pairing for Directing the Oxidative Folding of Peptides

Precise disulfide pairing in synthetic peptides usually is achieved using orthogonal protecting group strategies or relies on primary sequence manipulation. Orthogonal disulfide pairing technology should be promising for directing the rational folding of multicyclic peptides from the fully reduced peptides. Here, we report a discovery on the orthogonality between heterodisulfide pairing of cysteine (Cys) and penicillamine (Pen) and formation of Cys-Cys/Pen-Pen homodisulfides. The orthogonal Cys-Pen disulfide pairing can be exploited for highly selective production of certain (multi)­cyclic structures (or even a sole structure without isomers) through direct oxidation in air or thiol–disulfide exchanges in redox media. This strategy makes rational folding of multicyclic peptides without protecting groups, sequence manipulation, and complex synthetic reactions a reality, thus providing invaluable assets to peptide communities, and should greatly benefit the development of multicyclic peptide therapeutics and ligands

Fast and Selective Reaction of 2‑Benzylacrylaldehyde with 1,2-Aminothiol for Stable N‑Terminal Cysteine Modification and Peptide Cyclization

N-terminal cysteine (Cys)-specific reactions have been exploited for protein and peptide modifications. However, existing reactions for N-terminal Cys suffer from low reaction rate, unavoidable side reactions, or poor stability for reagents or products. Herein we report a fast, efficient, and selective conjugation between 2-benzylacrylaldehyde (BAA) and 1,2-aminothiol, which involves multistep reactions including aldimine condensation, Michael addition, and reduction of imine by NaBH3CN. This conjugation proceeds with a rate constant of ∼2700 M–1 s–1 under neutral condition at room temperature to produce a pair of seven-membered ring diastereoisomers, which are stable under neutral and acidic conditions. This method enables the selective modifications of the N-terminal Cys residue without interference from the internal Cys and lysine residues, providing a useful alternative to existing approaches for site-specific peptide or protein modifications and synthesis of cyclic peptides

Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers

The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity

Thioether-Bonded Fluorescent Probes for Deciphering Thiol-Mediated Exchange Reactions on the Cell Surface

Study on the processes of the thiol-mediated disulfide exchange reactions on the cell surface is not only important to our understanding of extracellular natural bioreduction processes but to the development of novel strategies for the intracellular delivery of synthetic bioactive molecules. However, disulfide-bonded probes have their intrinsic inferiority in exploring the detailed exchange pathway because of the bidirectional reactivity of disulfide bonds toward reactive thiols. In this work, we developed thioether-bonded fluorescent probes that enable us to explore thiol-mediated thioether (and disulfide) exchange reactions on the cell surface through fluorescence recovery and/or cell imaging. We demonstrated that our thioether-bonded probes can be efficiently cleaved through thiol-thioether exchanges with exofacial protein thiols and/or glutathione (GSH) efflux. The exchanges mainly take place on the cell surface, and GSH efflux-mediated exchange reactions can take place without the requirement of pre-exchanges of the probes with cell surface-associated protein thiols. On the basis of our founder methodology, for the first time we demonstrated the interplay of exofacial protein thiols and GSH efflux on the cleavage of external thioether-bonded compounds. Moreover, given that an understanding of the process of GSH efflux and the mechanism on which it relies is crucial to our understanding of the cellular redox homeostasis and the mechanism of multidrug resistance, we expect that our thioether-bonded probes and strategies would greatly benefit the fundamental study of GSH efflux in living cells

LSPR Sensing of Molecular Biothiols Based on Noncoupled Gold Nanorods

Au NRs protected with mPEG-SH molecules (mPEG-Au NRs) were demonstrated to be a promising platform for LSPR-based sensing of molecular biothiols in aqueous solution. Surface mPEG-SH molecules endow Au NRs with great stability and biocompatibility and no nonspecific adsorption of biomacromolecules. The LSPR band of mPEG-Au NRs displays a stability and linear response in the spectral shift with respect to a change in their surrounding refractive index with a sensitivity of 252 nm/RIU. The loose structure of mPEG-SH around the Au NRs offers free sites, thereby allowing molecular biothiols to bind onto the surfaces of Au NRs. The LSPR response and the sensitivity of Au NRs to biothiols such as GSH, Cys, Hcy, TGA, GSSG, and BSA were then studied

Table1_Genome-wide Identification and Expression Analysis of RcMYB Genes in Rhodiola crenulata.XLSX

Modern research has proved that the main medicinal component of Rhodiola crenulata, which has a wide range of medicinal value, is its secondary metabolite salidroside. The MYB transcription factor family is widely involved in biosynthesis of second metabolism and other roles in the stress response in plants, so a genome-wide identification and analysis for this family in R. crenulata is worth conducting. In this research, genome-wide analysis identified 139 MYB genes based on conserved domains in the R. crenulata genome, and 137 genes were used to construct a phylogenetic tree and modified with expression files to reveal evolutionary characteristics. Physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze RcMYBs. Additionally, cis-acting elements related to transcription, hormone, and MYB binding were found in the promoter region of the selected RcMYBs. Four RcMYBs were cloned, sequenced, and their gene expression pattern was analyzed for further analysis of their functions. The research results lay the foundation for further research on the function of RcMYB and R. crenulata.</p