Site-specific pegylated IL2 mutein with biased IL2 receptor binding for cancer immunotherapy.

While Interleukin 2 (IL2) has the capability to activate both NK and T cells robustly, its limited in vivo half-life, considerable toxicity, and tendency to boost Treg cells pose significant challenges, restricting its widespread application in cancer therapy. In this investigation, we engineered a novel IL2 variant (IL2-4M-PEG) with reduced CD25 binding activity and an extended half-life by substituting amino acids associated with CD25 binding and implementing site-directed PEGylation. IL2-4M-PEG notably amplifies effector cells over Treg cells. Furthermore, our findings reveal that IL2-4M-PEG, characterized by an extended half-life, exhibits anti-tumor effects in a mouse model. Consequently, this innovative IL2 holds the potential for enhancing combined cancer therapies in the future.

Protective effect of short-chain fructo-oligosaccharides from chicory on alcohol-induced injury in GES-1 cells via Keap1/Nrf2 and NLRP3 inflammasome signaling pathways.

Numerous studies have demonstrated that polysaccharides derived from chicory possess the ability to regulate host signaling and modify mucosal damage. Yet, the effect and mechanism of short-chain fructo-oligosaccharides (scFOS) on gastric mucosa remain unclear. Hence, the protective effect of three scFOS (1-Kestose, Nystose, and 1F-Fructofuranosylnystose) against ethanol-induced injury in gastric epithelial (GES-1) cells, and the underlying molecular mechanism involved was investigated in this study. Treatment with 7% ethanol decreased the cell viability of GES-1 cells, resulting in oxidative stress and inflammation. However, pretreatment with scFOS exhibited significant improvements in cell viability, and mitigated oxidative stress and inflammation. scFOS markedly elevated the protein expression of Nrf2, HO-1, SOD1 and SOD2, while suppressing the expression of Keap1. scFOS pretreatment could also maintain mitochondrial membrane potential balance and reduce apoptosis. In addition, scFOS was observed to reduce the protein level of NLRP3, Caspase-1 and ASC. In conclusion, scFOS served a preventive function in mitigating oxidative stress and inflammation in ethanol-exposed GES-1 cells through modulation of the Keap1/Nrf2 and NLRP3 inflammasome signaling pathways. Collectively, the results indicated that scFOS could significantly mitigate ethanol-induced gastric cell damage, suggesting its potential for safeguarding gastrointestinal health.

Phenolic acids from Chicory roots ameliorate dextran sulfate sodium-induced colitis in mice by targeting TRP signaling pathways and the gut microbiota.

BACKGROUND: Inflammatory bowel disease (IBD) is a type of immune-mediated condition associated with intestinal homeostasis. Our preliminary studies disclosed that Cichorium intybus L., a traditional medicinal plant, also known as Chicory in Western countries, contained substantial phenolic acids displaying significant anti-inflammatory activities. We recognized the potential of harnessing Chicory for the treatment of IBD, prompting a need for in-depth investigation into the underlying mechanisms. METHODS: On the third day, mice were given 100, 200 mg/kg of total phenolic acids (PA) from Chicory and 200 mg/kg of sulfasalazine (SASP) via gavage, while dextran sodium sulfate (DSS) concentration was 2.5 % for one week. The study measured and evaluated various health markers including body weight, disease activity index (DAI), colon length, spleen index, histological score, serum concentrations of myeloperoxidase (MPO), nitric oxide (NO), superoxide dismutase (SOD), lipid oxidation (MDA), and inflammatory factors. We evaluated the TRP family and the NLRP3 inflammatory signaling pathways by Western blot, while 16S rDNA sequencing was used to track the effects of PA on gut microbes. RESULTS: It was shown that PA ameliorated the weight loss trend, attenuated inflammatory damage, regulated oxidative stress levels, and repaired the intestinal barrier in DSS mice. Analyses of Western blots demonstrated that PA suppressed what was expressed of transient receptor potential family TRPV4, TRPA1, and the expression of NLRP3 inflammatory signaling pathway, NLRP3 and GSDMD. In addition, PA exerted therapeutic effects on IBD by regulating gut microbiota richness and diversity. Meanwhile, the result of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that gut microbiota was mainly related to Membrane Transport, Replication and Repair, Carbohydrate Metabolism and Amino Acid Metabolism. CONCLUSION: PA derived from Chicory may have therapeutic effects on IBD by regulating the TRPV4/NLRP3 signaling pathway and gut microbiome. This study provides new insights into the effects of phenolic acids from Chicory on TRP ion channels and gut microbiota, revealing previously unexplored modes of action.

One new 12, 8-guaianolide sesquiterpene lactone with antihyperglycemic activity from the roots of Cichorium intybus.

Three 12, 8-guaianolide sesquiterpene lactones, including a new compound intybusin F (1), and a new natural product cichoriolide I (2), along with six known 12, 6-guaianolide compounds (4-9) were isolated from the roots of Cichorium intybus L. Their structures were determined by extensive spectroscopic analysis. The absolute configurations of new compounds were elucidated based on analysis of the experimental and calculated electronic circular dichroism spectra. Compounds 1, 2, 4, 7, 8 showed significant effects on facilitating the glucose uptake in oleic acid plus high glucose-stimulated HepG2 cells at 50 µM. In addition, compounds 1, 2, 3, 6, 7 exhibited obvious inhibitory effects against NO production, of them, compounds 1, 2, 7 can significantly decrease the secretion of inflammatory cytokines (TNF-α, IL-6 and COX-2) levels in this hyperglycemic HepG2 cell model.

Microbiota and plant-derived vesicles that serve as therapeutic agents and delivery carriers to regulate metabolic syndrome.

The gut is a fundamental organ in controlling human health. Recently, researches showed that substances in the intestine can alter the course of many diseases through the intestinal epithelium, especially intestinal flora and exogenously ingested plant vesicles that can be transported over long distances to various organs. This article reviews the current knowledge on extracellular vesicles in modulating gut homeostasis, inflammatory response and numerous metabolic disease that share obesity as a co-morbidity. These complex systemic diseases that are difficult to cure, but can be managed by some bacterial and plant vesicles. Vesicles, due to their digestive stability and modifiable properties, have emerged as novel and targeted drug delivery vehicles for effective treatment of metabolic diseases.

Total Sesquiterpenoids of Loquat Leaves Alleviated High-Fat Diet-Induced Obesity by Targeting Fecal Metabolic Profiling and Gut Microbiota Composition.

In the present study, we demonstrated that whether the gut microbiota and related metabolites contribute to the therapeutic effect of total sesquiterpenoids (TSs) from loquat leaves on obesity. A 4-week high fat diet was used to induce obesity which was then treated with TSs for another 4 weeks. TSs remarkedly reduced the weight of body and white adipose and the levels of total cholesterol (TC) and triglyceride (TG) in serum. We also found that TSs restored the diversity and richness of gut microbiota. In addition, TSs administration affected the relative abundance of seven key genera. Meanwhile, TSs were determined to affect the metabolism of the host through detecting the metabolites in feces. By applying KEGG and the correlation analysis with gut microbiota, 10 differential metabolites were identified to be the key. The results in this work proved that TSs inhibited obesity by remodeling gut microbiota and related metabolites.

Chicoric Acid Attenuated Renal Tubular Injury in HFD-Induced Chronic Kidney Disease Mice through the Promotion of Mitophagy via the Nrf2/PINK/Parkin Pathway.

As the main factor in the pathogenesis of chronic kidney disease (CKD), the excessive apoptosis of renal tubular epithelial cells (RTECs) and its underlying mechanism of action are worth further investigation. Chicoric acid (CA), a major active constituent of the Uyghur folk medicine chicory, was recorded to possess a renal protective effect. The precise effect of CA on renal tubular injury in obesity-related CKD remains unknown. In the current study, CA was proven to ameliorate metabolic disorders including overweight, hyperglycemia, hyperlipidemia, and hyperuricemia in high fat diet (HFD)-fed mice. Furthermore, the reverse effect of CA on renal histological changes and functional damage was confirmed. In vitro, the alleviation of lipid accumulation and cell apoptosis was observed in palmitic acid (PA)-exposed HK2 cells. Treatment with CA reduced mitochondrial damage and oxidative stress in the renal tubule of HFD-fed mice and PA-treated HK2 cells. Finally, CA was observed to activate the Nrf2 pathway; increase PINK and Parkin expression; and regulate LC3, SQSTM1, Mfn2, and FIS1 expression; therefore, it would improve mitochondrial dynamics and mitophagy to alleviate mitochondrial damage in RTECs of obesity-related CKD. These results may provide fresh insights into the promotion of mitophagy in the prevention and alleviation of obesity-related CKD.

Comparative Label-Free Quantitative Proteomics Analysis Reveals the Essential Roles of N-Glycans in Salt Tolerance by Modulating Protein Abundance in Arabidopsis.

Many pieces of evidence show that the adaptive response of plants to salt stress requires the maturation of N-glycan on associated proteins. However, it is still little known about the salt-responsive glycoproteins that function in this process. In the present study, we identified salt-responsive glycoproteins in wild-type (WT) Arabidopsis and two mutants defective in N-glycan maturation, mns1 mns2 and cgl1. A total of 97 proteins with abundance changes of >1.5- or <0.67-fold were identified against salt stress by label-free liquid chromatography coupled mass spectrometry (LC-MS/MS) quantitative analyses. A comparison of differentially abundant glycoproteins (DAGs) indicated the substrate preferences regulated by MNS1/MNS2 and CGL1. In addition, the DAGs in mns1 mns2 hardly form functional regulatory networks in STRING analysis. Comparably, the regulatory network in cgl1 was visible and shared overlapping with that in WT. Such difference may supply the evidence to partially explain the lower salt sensitivity of mutant cgl1 than mns1 mns2. We further confirmed that two N-glycosylation clients, peroxidases PRX32 and PRX34, were involved in the salt stress response since the double mutants showed enhanced salt sensitivity. Together, our study provided proteomic evidence that N-glycans are crucial for modulating stress-responsive protein levels, and several novel glycoproteins responsible for salt stress tolerance in Arabidopsis were listed. Data are available via ProteomeXchange with identifier PXD006893.

N-glycosylation is involved in stomatal development by modulating the release of active abscisic acid and auxin in Arabidopsis.

Asparagine-linked glycosylation (N-glycosylation) is one of the most important protein modifications in eukaryotes, affecting the folding, transport, and function of a wide range of proteins. However, little is known about the roles of N-glycosylation in the development of stomata in plants. In the present study, we provide evidence that the Arabidopsis stt3a-2 mutant, defective in oligosaccharyltransferase catalytic subunit STT3, has a greater transpirational water loss and weaker drought avoidance, accompanied by aberrant stomatal distribution. Through physiological, biochemical, and genetic analyses, we found that the abnormal stomatal density of stt3a-2 was partially attributed to low endogenous abscisic acid (ABA) and auxin (IAA) content. Exogenous application of ABA or IAA could partially rescue the mutant's salt-sensitive and abnormal stomatal phenotype. Further analyses revealed that the decrease of IAA or ABA in stt3a-2 seedlings was associated with the underglycosylation of ß-glucosidase (AtBG1), catalysing the conversion of conjugated ABA/IAA to active hormone. Our results provide strong evidence that N-glycosylation is involved in stomatal development and participates in abiotic stress tolerance by modulating the release of active plant hormones.

Multiple N-glycans cooperate in balancing misfolded BRI1 secretion and ER retention.

KEY MESSAGE: N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.

The Evolutionarily Conserved Serine Residues in BRI1 LRR Motifs Are Critical for Protein Secretion.

As a well-studied leucine-rich-repeat receptor-like kinases (LRR-RLKs) in Arabidopsis (Arabidopsis thaliana), BRI1 functions as a cell surface receptor for sensing the smallest ligand molecule identified thus far. The weak allele bri1-9 (S662F) harbors a mutation at the conserved serine (Ser*) residue among 25 LRRs, which leads to the protein retention in the ER. However, very little is known about the importance of these residues. Through site-directed mutagenesis and a phenotypic complementation test, we examined the effects of these conserved serine residues (S*-chain) on protein secretion and functions. The results showed that the replacements of these serine residues significantly changed the sub-localization of BRI1-GFPs to the ER and that rigid space constraints, as well as the requirement of successive inner polar contacts, affect these sites. In addition, the continuous presence of Ser* is mainly disrupted at the LRR-island domain interface, and the changes of these four nonserine residues to serine greatly decreased the protein ability to complement bri1-301 compact phenotype and the BR signaling activation. The sequence alignment revealed that other known LRR-RLK also harbors the S*-chain and the non-Ser* residues at the ligand-binding region along the S*-chain, which confirms the evolutionary significance of residues at these sites in plant LRR-RLKs.

Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms.

STT3 is a catalytic subunit of hetero-oligomeric oligosaccharyltransferase (OST), which is important for asparagine-linked glycosylation. In mammals and plants, OSTs with different STT3 isoforms exhibit distinct levels of enzymatic efficiency or different responses to stressors. Although two different STT3 isoforms have been identified in both plants and animals, it remains unclear whether these isoforms result from gene duplication in an ancestral eukaryote. Furthermore, the molecular mechanisms underlying the functional divergences between the two STT3 isoforms in plant have not been well elucidated. Here, we conducted phylogenetic analysis of the major evolutionary node species and suggested that gene duplications of STT3 may have occurred independently in animals and plants. Across land plants, the exon-intron structure differed between the two STT3 isoforms, but was highly conserved for each isoform. Most angiosperm STT3a genes had 23 exons with intron phase 0, while STT3b genes had 6 exons with intron phase 2. Characteristic motifs (motif 18 and 19) of STT3s were mapped to different structure domains in the plant STT3 proteins. These two motifs overlap with regions of high nonsynonymous-to-synonymous substitution rates, suggesting the regions may be related to functional difference between STT3a and STT3b. In addition, promoter elements and gene expression profiles were different between the two isoforms, indicating expression pattern divergence of the two genes. Collectively, the identified differences may result in the functional divergence of plant STT3s.

Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis.

Asparagine (Asn/N)-linked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mns1 mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mns1 mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-ß-1,4-endoglucanase (RSW2/KOR) that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mns1 mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.