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Biomedicine & Pharmacotherapy =... Apr 2024Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and...
Koumine inhibits IL-1β-induced chondrocyte inflammation and ameliorates extracellular matrix degradation in osteoarthritic cartilage through activation of PINK1/Parkin-mediated mitochondrial autophagy.
Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and treatment of osteoarthritis. Koumine is a bioactive alkaloid extracted from the plant Gelsemium elegans. In previous research, Koumine was found to have potential in improving the progression of OA in rats. However, the specific mechanism of its action has not been fully explained. Therefore, the aim of this study was to investigate whether Koumine can alleviate OA in rats by influencing mitochondrial autophagy. In the in vitro study, rat chondrocytes (RCCS-1) were induced with IL-1β (10 ng/mL) to induce inflammation, and Koumine (50 μg/mL) was co-treated. In the in vivo study, a rat OA model was established by intra-articular injection of 2% papain, and Koumine was administered orally (1 mg/kg, once daily for two weeks). It was found that Koumine effectively reduced cartilage erosion in rats with osteoarthritis. Additionally, it decreased the levels of inflammatory factors such as IL-1β, IL-6, and extracellular matrix (ECM) components MMP13 and ADAMTS5 in chondrocytes and articular cartilage tissue, while increasing the level of Collagen II.Koumine inhibited the production of reactive oxygen species (ROS) in cartilage tissue and increased the number of autophagosomes in chondrocytes and articular cartilage tissue. Additionally, it upregulated the expression of mitochondrial autophagy proteins LC3Ⅱ/Ⅰ, PINK1, Parkin, and Drp1. The administration of Mdivi-1 (50 μM) reversed the enhanced effect of Koumine on mitochondrial autophagy, as well as its anti-inflammatory and anti-ECM degradation effects in rats with OA. These findings suggest that Koumine can alleviate chondrocyte inflammation and improve the progression of OA in rats by activating PINK1/Parkin-mediated mitochondrial autophagy.
Topics: Rats; Animals; Chondrocytes; Osteoarthritis; Rats, Sprague-Dawley; Inflammation; Cartilage, Articular; Autophagy; Interleukin-1beta; Extracellular Matrix; Ubiquitin-Protein Ligases; Protein Kinases; Indole Alkaloids
PubMed: 38412715
DOI: 10.1016/j.biopha.2024.116273 -
Nature Plants Apr 2024Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as...
Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.
Topics: Oryza; Plant Diseases; Fungal Proteins; Plant Proteins; Host-Pathogen Interactions; Papain; Ascomycota; Magnaporthe
PubMed: 38409290
DOI: 10.1038/s41477-024-01642-x -
Pharmaceutics Jan 2024The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic...
The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PL), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PL, with an IC50 value of 82 ± 34 μM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PL, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PL inhibitor development, this study elaborates on the PL binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PL inhibitor development.
PubMed: 38399230
DOI: 10.3390/pharmaceutics16020169 -
Applied Microbiology and Biotechnology Feb 2024Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the...
Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. KEY POINTS: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.
Topics: Swine; Animals; Papain; Pork Meat; Red Meat; Fatty Acids, Unsaturated; Linoleic Acid; Oxygen; Mucor
PubMed: 38376614
DOI: 10.1007/s00253-024-13018-4 -
Frontiers in Plant Science 2024In plants, serpins are a superfamily of serine and cysteine protease inhibitors involved in stress and defense mechanisms, with potential for controlling agricultural...
In plants, serpins are a superfamily of serine and cysteine protease inhibitors involved in stress and defense mechanisms, with potential for controlling agricultural pests, making them important biotechnological tools. The objective of this study was to characterize a serpin from , called TcSERPIN, to identify its endogenous targets and determine its function and biotechnological potential. TcSERPIN has 390 amino acid residues and shows conservation of the main active site, RCL. Cis-elements related to light, stress, hormones, anaerobic induction, cell cycle regulation and defense have been identified in the gene's regulatory region. TcSERPIN transcripts are accumulated in different tissues of . Furthermore, in plants infected with and , the expression of TcSERPIN was positively regulated. The protein spectrum, rTcSERPIN, reveals a typical β-sheet pattern and is thermostable at pH 8, but loses its structure with temperature increases above 66°C at pH 7. At the molar ratios of 0.65 and 0.49, rTcSERPIN inhibited 55 and 28% of the activity of papain from and trypsin from , respectively. The protease trap containing immobilized rTcSERPIN captured endogenous defense proteins from cocoa extracts that are related to metabolic pathways, stress and defense. The evaluation of the biotechnological potential against geohelminth larvae showed that rTcSERPIN and rTcCYS4 ( cystatin 4) reduced the movement of larvae after 24 hours. The results of this work show that TcSERPIN has ideal biochemical characteristics for biotechnological applications, as well as potential for studies of resistance to phytopathogens of agricultural crops.
PubMed: 38348273
DOI: 10.3389/fpls.2024.1337750 -
Molecules (Basel, Switzerland) Feb 2024This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit () through ethanol precipitation at different ratios, and to determine its proteolytic...
This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit () through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation ( < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.
Topics: Animals; Humans; Hydrolysis; Bombyx; Papain; Fruit; Powders; Peptide Hydrolases; Whey Proteins; Maclura; Soybean Proteins; Subtilisins; Ethanol
PubMed: 38338437
DOI: 10.3390/molecules29030693 -
Nutrients Jan 2024The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates...
Olive ( L.) Seed as New Source of Cholesterol-Lowering Bioactive Peptides: Elucidation of Their Mechanism of Action in HepG2 Cells and Their Trans-Epithelial Transport in Differentiated Caco-2 Cells.
The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.
Topics: Humans; Hep G2 Cells; Proprotein Convertase 9; Caco-2 Cells; Olea; Peptides; Cholesterol; Seeds
PubMed: 38337656
DOI: 10.3390/nu16030371 -
RSC Chemical Biology Feb 2024The SARS-CoV-2 papain-like protease (PL) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural...
The SARS-CoV-2 papain-like protease (PL) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PL. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PL. The results reveal how the substrate sequence affects the efficiency of PL-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PL catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PL. The results will help in identifying PL substrates and guiding inhibitor design.
PubMed: 38333195
DOI: 10.1039/d3cb00128h -
Journal of Orthopaedic Surgery and... Feb 2024Osteoarthritis (OA) is a degenerative joint disease caused by the deterioration of cartilage. However, the underlying mechanisms of OA pathogenesis remain elusive.
BACKGROUND
Osteoarthritis (OA) is a degenerative joint disease caused by the deterioration of cartilage. However, the underlying mechanisms of OA pathogenesis remain elusive.
METHODS
Hub genes were screened by bioinformatics analysis based on the GSE114007 and GSE169077 datasets. The Sprague-Dawley (SD) rat model of OA was constructed by intra-articular injection of a mixture of papain and L-cysteine. Hematoxylin-eosin (HE) staining was used to detect pathological changes in OA rat models. Inflammatory cytokine levels in serum were measured employing the enzyme-linked immunosorbent assay (ELISA). The reverse transcription quantitative PCR (RT-qPCR) was implemented to assess the hub gene expressions in OA rat models. The roles of PDK4 and the mechanism regulating the PPAR pathway were evaluated through western blot, cell counting kit-8 (CCK-8), ELISA, and flow cytometry assays in C28/I2 chondrocytes induced by IL-1β.
RESULTS
Six hub genes were identified, of which COL1A1, POSTN, FAP, and CDH11 expressions were elevated, while PDK4 and ANGPTL4 were reduced in OA. Overexpression of PDK4 inhibited apoptosis, inflammatory cytokine levels (TNF-α, IL-8, and IL-6), and extracellular matrix (ECM) degradation protein expressions (MMP-3, MMP-13, and ADAMTS-4) in IL-1β-induced chondrocytes. Further investigation revealed that PDK4 promoted the expression of PPAR signaling pathway-related proteins: PPARA, PPARD, and ACSL1. Additionally, GW9662, an inhibitor of the PPAR pathway, significantly counteracted the inhibitory effect of PDK4 overexpression on IL-1β-induced chondrocytes.
CONCLUSION
PDK4 inhibits OA development by activating the PPAR pathway, which provides new insights into the OA management.
Topics: Rats; Animals; Peroxisome Proliferator-Activated Receptors; Osteoarthritis; Cells, Cultured; Rats, Sprague-Dawley; Chondrocytes; Cytokines; Interleukin-1beta; Inflammation
PubMed: 38308345
DOI: 10.1186/s13018-024-04583-5 -
Nature Communications Feb 2024
PubMed: 38307911
DOI: 10.1038/s41467-024-45406-9