An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked ß-N-Acetylglucosamine Strategy.

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked ß-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor ß2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

The quality of life after keratinized mucosa augmentation around dental implants using xenogenic collagen matrix with or without stent.

BACKGROUND: The substitution of missing teeth with implants is a dependable and anticipated therapeutic approach. Despite numerous studies affirming long-term success rates, there exists a spectrum of potential biological and aesthetic complications. OBJECTIVE: The primary objective of this study was to assess patient responses subsequent to surgical interventions, with a specific emphasis on the utilization of xenogenic collagen matrix (XCM), both with and without the application of a stent secured over healing abutments, in the context of keratinized gingival mucosa augmentation. The principal aim was to evaluate and draw comparisons between the clinical outcomes resulting from these two procedural approaches, with a particular focus on critical parameters encompassing post-operative complications, patient comfort, and the overall efficacy in achieving successful keratinized tissue augmentation. methods: Sixty patients were selected for this study. The patients were divided into three groups: A, B, and a control group, with each group comprising 20 participants. We used XCM in experimental group A, XCM covered with surgical stent in experimental group B, and free gingival graft (FGG) in the control group. After the surgical procedure, patients were required to complete a visual analogue scale (VAS) questionnaire for post-operative complications, and a quality of life (QOL) questionnaire on days 1, 3, and 7. RESULTS: Patients in the experimental groups A and B demonstrated markedly improved outcomes when compared with the control group. Assessments conducted on days 1, 3, and 7 demonstrated diminished levels of pain, bleeding, and swelling in both experimental groups, with experimental group B showing the least discomfort. The incorporation of XCM, either with or without stents, was associated with a reduction in analgesic consumption, underscoring its favorable influence on post-operative comfort, notwithstanding the exception of halitosis in experimental group B. CONCLUSION: Using XCM with or without a stent for keratinized tissue augmentation has better post-operative outcomes associated with reduced swelling, bleeding, and pain based on the QOL survey. This study provides data to support the clinical application of XCM and stents.

Mega-oss and Mega-TCP versus Bio-Oss granules fixed by alginate gel for bone regeneration.

OBJECTIVES/AIMS: Bone graft materials are widely used at present because inadequate bone volume is usually found in implant patients. To determine the biocompatibility of a new grafting material, in vitro research is routinely performed before animal experiments and clinical testing. However, during in vitro experiments, bone material particles might move during testing, which could affect the accuracy of the results. MATERIALS AND METHODS: To evaluate the biocompatibility of new bone substitutes, Mega-oss and Mega-TCP were compared with Bio-Oss using osteoblast cells and osteoclast cells fixed with alginate gel. Cell morphology, viability, bone resorption, alkaline phosphatase (ALP) activity, and staining were tested to compare the biocompatibility differences in the performance of Mega-oss, Mega-TCP, and Bio-Oss. RESULTS: Cells spread better on Mega-oss and Mega-TCP than the round shape on Bio-Oss. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) results showed that Mega-oss, Mega-TCP, and sodium alginate had good viability. Meanwhile, Mega-oss and Mega-TCP had the same osteoblast differentiation ability as Bio-Oss. The resorption rates of Mega-TCP and Mega-oss were higher than those of Bio-Oss (24.4%, 15.3%, and 3.3%, respectively). CONCLUSION: Mega-oss and Mega-TCP might be useful alternative bone graft materials compared with Bio-Oss. In addition, fixing the materials with sodium alginate gel could be a new method for in vitro bone material experiments.