Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute a vast and diverse family of bioactive peptides. These peptides, synthesized by ribosomes and subsequently modified by various tailoring enzymes, possess a wide chemical space. Among these modifications, radical S-adenosylmethionine (rSAM) enzymes employ unique radical chemistry to introduce a variety of novel peptide structures, which are crucial for their activity. This review examines the major types of modifications in RiPPs catalyzed by rSAM enzymes, incorporating recent advancements in protein structure analysis techniques and computational methods. Additionally, it elucidates the diverse catalytic mechanisms and substrate selectivity of these enzymes through an analysis of the latest crystal structures.
Protein Processing, Post-Translational ; S-Adenosylmethionine/chemistry* ; Ribosomes/metabolism* ; Peptides/metabolism* ; Biological Products/metabolism* ; Humans

OBJECTIVES: This study investigated the effects of a polycaprolactone (PCL)-polyethylene glycol (PEG) scaffold incorporated with concentrated growth factor (CGF) on the adhesion, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). METHODS: The PCL-PEG-CGF composite scaffold was fabricated using an immersion and freeze-drying technique. Its microstructure, mechanical properties, and biocompatibility were systematically characterized. The hPDLSCs were isolated through enzymatic digestion, and the hPDLSCs were identified through flow cytometry. Third-passage hPDLSCs were seeded onto the composite scaffolds, and their adhesion, proliferation and osteogenic differentiation were assessed using CCK-8 assays, 4',6-diamidino-2-phenylindole (DAPI) staining, alkaline phosphatase (ALP) staining, alizarin red staining, and Western blot analysis of osteogenesis-related proteins [Runt-related transcription factor 2 (Runx2), ALP, and morphogenetic protein 2 (BMP2)]. RESULTS: Scanning electron microscopy revealed that the PCL-PEG-CGF composite scaffold exhibited a honeycomb-like structure with heterogeneous pore sizes. The composite scaffold exhibited excellent hydrophilicity, as evidenced by a contact angle (θ) approaching 0° within 6 s. Its elastic modulus was measured at (4.590 0±0.149 3) MPa, with comparable hydrophilicity, fracture tensile strength, and fracture elongation to PCL-PEG scaffold. The hPDLSCs exhibited significantly improved adhesion to the PCL-PEG-CGF composite scaffold compared with the PCL-PEG scaffold (P<0.01). Additionally, cell proliferation was markedly improved in all the experimental groups on days 3, 5, and 7 (P<0.01), and statistically significant differences were found between the PCL-PEG-CGF group and other groups (P<0.01). The PCL-PEG-CGF group showed significantly elevated ALP activity (P<0.05), increased mineralization nodule formation, and upregulated expression of osteogenic-related proteins (Runx2, BMP2 and ALP; P<0.05). CONCLUSIONS The PCL-PEG-CGF composite scaffold exhibited excellent mechanical properties and biocompatibility, enhancing the adhesion and proliferation of hPDLSCs and promoting their osteogenic differentiation by upregulating osteogenic-related proteins.
Humans ; Polyesters/chemistry* ; Periodontal Ligament/cytology* ; Polyethylene Glycols/chemistry* ; Stem Cells/cytology* ; Tissue Scaffolds ; Cell Proliferation ; Osteogenesis ; Cell Differentiation ; Cell Adhesion ; Bone Morphogenetic Protein 2/metabolism* ; Cells, Cultured ; Alkaline Phosphatase/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Intercellular Signaling Peptides and Proteins/pharmacology* ; Tissue Engineering/methods*

OBJECTIVE: To review the research progress on bone repair biomaterials with the function of recruiting endogenous mesenchymal stem cells (MSCs). METHODS: An extensive review of the relevant literature on bone repair biomaterials, particularly those designed to recruit endogenous MSCs, was conducted, encompassing both domestic and international studies from recent years. The construction methods and optimization strategies for these biomaterials were summarized. Additionally, future research directions and focal points concerning this material were proposed. RESULTS: With the advancement of tissue engineering technology, bone repair biomaterials have increasingly emerged as an ideal solution for addressing bone defects. MSCs serve as the most critical "seed cells" in bone tissue engineering. Historically, both MSCs and their derived exosomes have been utilized in bone repair biomaterials; however, challenges such as limited sources of MSCs and exosomes, low survival rates, and various other issues have persisted. To address these challenges, researchers are combining growth factors, bioactive peptides, specific aptamers, and other substances with biomaterials to develop constructs that facilitate stem cell recruitment. By optimizing mechanical properties, promoting vascular regeneration, and regulating the microenvironment, it is possible to create effective bone repair biomaterials that enhance stem cell recruitment. CONCLUSION In comparison to cytokines, phages, and metal ions, bioactive peptides and aptamers obtained through screening exhibit more specific and targeted recruitment functions. Future development directions for bone repair biomaterials will involve the modification of peptides and aptamers with targeted recruitment capabilities in biological materials, as well as the optimization of the mechanical properties of these materials to enhance vascular regeneration and adjust the microenvironment.
Mesenchymal Stem Cells/metabolism* ; Biocompatible Materials/chemistry* ; Tissue Engineering/methods* ; Humans ; Bone Regeneration ; Tissue Scaffolds/chemistry* ; Animals ; Bone and Bones ; Mesenchymal Stem Cell Transplantation/methods* ; Exosomes/metabolism* ; Intercellular Signaling Peptides and Proteins/metabolism* ; Osteogenesis

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.
Metallothionein/metabolism* ; Elastin/chemistry* ; Recombinant Fusion Proteins/pharmacology* ; Mice ; Animals ; Peptides/metabolism* ; Escherichia coli/metabolism* ; NIH 3T3 Cells

Antimicrobial peptides (AMPs) are small molecular peptides widely existing in the innate immunity of organisms, serving as the first line of defense. Natural AMPs possess various biological activities and are difficult to develop drug resistance. However, they are easily broken down by digestive enzymes in the body. In recent years, increasing methods have been reported to enhance the stability of AMPs, including incorporation of unnatural amino acids, chemical modifications, strategic avoidance of enzyme cleavage sites, cyclization, and nano peptide design. This review summarizes the methods for improving the stability of AMPs against protease degradation, aiming to provide references for further research in this field.
Antimicrobial Peptides/pharmacology* ; Humans ; Peptide Hydrolases/metabolism* ; Protein Stability ; Antimicrobial Cationic Peptides/chemistry* ; Anti-Infective Agents/chemistry*

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.
AMP-Activated Protein Kinases/metabolism* ; Alzheimer Disease/drug therapy* ; Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Atractylodes/chemistry* ; Autophagy/drug effects* ; Beclin-1/pharmacology* ; Caenorhabditis elegans/metabolism* ; Macroautophagy ; Mice ; RNA, Messenger ; Sirolimus/pharmacology*

Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)_n. The pET28a-Trxm-(TRSP)_n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)_n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)₁ achieved 50% of the total protein, while the expression level of Trxm-(TRSP)₂ was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)₁ were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.
Escherichia coli/metabolism* ; Peptides/chemistry* ; Amino Acid Sequence ; Gene Library ; Tandem Repeat Sequences

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.
Berberidaceae ; microbiology ; Endophytes ; Fungi ; chemistry ; HIV Integrase ; metabolism ; HIV-1 ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Protein Binding

The aim of this paper was to discuss the protective effect and mechanism of Acanthopanax senticosus polysaccharides( ASPs) on immunological liver injury caused by conanavalin A( Con A). BALB/c mice were randomly divided into seven groups: control group,model group( Con A),low-,medium-,and high-dose( 36. 25,72. 5,145 mg·kg~(-1)) ASPs groups,bifendate( 200 mg·kg~(-1),positive drug) group and pyrrolidinedithiocarbamate( PDTC,NF-κB inhibitor,200 mg·kg~(-1)) group. ASPs groups and bifendate group were given with corresponding drugs by ig administration once daily for 7 d. Control group,model group and PDTC group were given with normal saline by ig administration once daily for 7 d. After the last ig administration,PDTC was given in DTC group by iv administration( 200 mg·kg~(-1)); 0. 5 h after that,Con A( 20 mg·kg~(-1)) was injected via the tail vein to induce immunological liver injury in all the mice except normal control group. The mice were killed 8 h later and their liver tissues were collected for histopathological examination. The contents of nitric oxide( NO),superoxide dismutase( SOD),malondialdehyde( MDA),reduced glutathione( GSHPX),interleukin( IL-1β) and tumor necrosis factor( TNF-α) in liver tissues were detected by kit assay. Western blot method was used to detect TNF-α,intercellular cell adhesion molecule-1( ICAM-1),inducible nitric oxide synthase( i NOS) and nuclear factor( NF-κB) protein expression in liver tissues. As compared with model group,ASPs not only could reduce the activity of MDA,NO,IL-1β and TNF-α,but also increase the content of GSH-PX and SOD; at the same time,the protein expression levels of TNF-α,ICAM-1,i NOS and NF-κB were reduced in liver tissues; in addition,inflammatory cell infiltration was alleviated,hepatocyte cytoplasm was loose and swollen,and nuclear condensation and staining were improved. ASPs has a protective effect on immunological liver injury,and the mechanism may be associated with regulating secretion of inflammatory cytokines and the expression of adhesion factor through NF-κB signaling pathway.
Animals ; Chemical and Drug Induced Liver Injury ; drug therapy ; Cytokines ; metabolism ; Eleutherococcus ; chemistry ; Liver ; drug effects ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; metabolism ; Peptides, Cyclic ; Polysaccharides ; pharmacology ; Random Allocation ; Signal Transduction

Romipeptides A and B (1 and 2), two new romidepsin derivatives, and three known compounds, chromopeptide A (3), romidepsin (4) and valine-leucine dipeptide (5) were isolated from the fermentation broth of Chromobacterium violaceum No. 968. Their structures were elucidated by interpretation of their UV, HR-ESI-MS and NMR spectra. The absolute configuration of compound 1 and 2 were established by single crystal X-ray diffraction analysis. Compounds 1-5 were evaluated for their anti-proliferative activities against three human cancer cell lines, SW620, HL60, and A549. The results showed most of these compounds exhibited antitumor activities in vitro, in which compound 2 displayed potent cytotoxicity to SW620, HL60 and A549 cell lines, with IC of 12.5, 6.7 and 5.7 nmol·L, respectively.
Antineoplastic Agents ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Chemistry Techniques, Analytical ; Chromobacterium ; metabolism ; Depsipeptides ; chemistry ; pharmacology ; Dipeptides ; chemistry ; Drug Screening Assays, Antitumor ; Fermentation ; Humans ; Molecular Structure ; Peptides, Cyclic ; chemistry