bims-supasi 2024-05-26 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2024‒05‒26
fifteen papers selected by
Jonathan Wolf Mueller, University of Birmingham

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.
Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.
Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.
Targeting chondroitin sulfate suppresses macropinocytosis of breast cancer cells by modulating syndecan-1 expression.
Sex-specific association of serum dehydroepiandrosterone and its sulfate levels with osteoporosis in type 2 diabetes.
Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease.
Chondroitin sulfate-based universal nanoparticle delivers angiogenic inhibitor and paclitaxel to exhibit a combination of chemotherapy and anti-angiogenic therapy.
Identification and characterization of a chondroitinase ABC with a novel carbohydrate-binding module.
Versican controlled by Lmx1b regulates hyaluronate density and hydration for semicircular canal morphogenesis.
Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.
Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.
Sulfate glycosaminoglycan from swim bladder exerts immunomodulatory potential on macrophages via toll-like receptor 4 mediated NF-κB signaling pathways.
Renoprotective effect of rosmarinic acid by inhibition of indoxyl sulfate-induced renal interstitial fibrosis via the NLRP3 inflammasome signaling.
Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing.
Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

Angew Chem Int Ed Engl. 2024 May 23. e202405671

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Po-Han Lin, Yongmei Xu, Semiha Kevser Bali, Jandi Kim, Ana Gimeno, Elijah T Roberts, Deepak James, Nuno M S Almeida, Narasimhan Loganathan, Fei Fan, Angela K Wilson, I Jonathan Amster, Kelley W Moremen, Jian Liu, Jesús Jiménez-Barbero, Xuefei Huang.

  Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

Keywords:  Chondroitin sulfate glycopeptide; enzymes; proteoglycan; solid phase; synthesis

DOI:  https://doi.org/10.1002/anie.202405671
Mar Drugs. 2024 Apr 26. pii: 198. [Epub ahead of print]22(5):

Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.

Zhe Wang, Weiwen Wang, Hao Gong, Yudi Jiang, Renjie Liu, Guangli Yu, Guoyun Li, Chao Cai.

  Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 μg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.

Keywords:  HPLC-MS/MS; flounder; glycosaminoglycan; structure properties

DOI:  https://doi.org/10.3390/md22050198
Mar Drugs. 2024 May 20. pii: 232. [Epub ahead of print]22(5):

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Jiyuan Yang, Yuefan Song, Ke Xia, Vitor H Pomin, Chunyu Wang, Mingqiang Qiao, Robert J Linhardt, Jonathan S Dordick, Fuming Zhang.

  Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Keywords:  HSPGs; Mycoplasma pneumoniae; heparin; marine sulfated glycans; surface plasmon resonance

DOI:  https://doi.org/10.3390/md22050232
Mol Oncol. 2024 May 21.

Targeting chondroitin sulfate suppresses macropinocytosis of breast cancer cells by modulating syndecan-1 expression.

Hung-Rong Yen, Wen-Chieh Liao, Chia-Hua Chen, Ying-Ai Su, Ying-Wei Huang, Chi Hsiao, Yu-Lun Chou, Yin-Hung Chu, Pin-Keng Shih, Chiung-Hui Liu.

  Accumulation of abnormal chondroitin sulfate (CS) chains in breast cancer tissue is correlated with poor prognosis. However, the biological functions of these CS chains in cancer progression remain largely unknown, impeding the development of targeted treatment focused on CS. Previous studies identified chondroitin polymerizing factor (CHPF; also known as chondroitin sulfate synthase 2) is the critical enzyme regulating CS accumulation in breast cancer tissue. We then assessed the association between CHPF-associated proteoglycans (PGs) and signaling pathways in breast cancer datasets. The regulation between CHPF and syndecan 1 (SDC1) was examined at both the protein and RNA levels. Confocal microscopy and image flow cytometry were employed to quantify macropinocytosis. The effects of the 6-O-sulfated CS-binding peptide (C6S-p) on blocking CS functions were tested in vitro and in vivo. Results indicated that the expression of CHPF and SDC1 was tightly associated within primary breast cancer tissue, and high expression of both genes exacerbated patient prognosis. Transforming growth factor beta (TGF-β) signaling was implicated in the regulation of CHPF and SDC1 in breast cancer cells. CHPF supported CS-SDC1 stabilization on the cell surface, modulating macropinocytotic activity in breast cancer cells under nutrient-deprived conditions. Furthermore, C6S-p demonstrated the ability to bind CS-SDC1, increase SDC1 degradation, suppress macropinocytosis of breast cancer cells, and inhibit tumor growth in vivo. Although other PGs may also be involved in CHPF-regulated breast cancer malignancy, this study provides the first evidence that a CS synthase participates in the regulation of macropinocytosis in cancer cells by supporting SDC1 expression on cancer cells.

Keywords:  CHPF; breast cancer; chondroitin sulfate; macropinocytosis; syndecan 1

DOI:  https://doi.org/10.1002/1878-0261.13667
J Bone Miner Metab. 2024 May 20.

Sex-specific association of serum dehydroepiandrosterone and its sulfate levels with osteoporosis in type 2 diabetes.

Shuo Li, Wei Li, Lina Chang, Jieying Wan, Shanshan Chen, Xinxin Zhang, Qing He, Ming Liu.

  INTRODUCTION: This study is to investigate the relation between serum dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) levels and the risk of osteoporosis in patients with T2DM.MATERIALS AND METHODS: This cross-sectional study involved 938 hospitalized patients with T2DM. Linear regression models were used to explore the relationship between DHEA and DHEAS and the BMD at different skeletal sites. Multinominal logistic regression models and the restricted cubic spline (RCS) were used to evaluate the associations of DHEA and DHEAS with the risks of osteopenia and/or osteoporosis.
RESULTS: In postmenopausal women with T2DM, after adjustment for confounders including testosterone and estradiol, DHEA showed a significant positive correlation with lumbar spine BMD (P = 0.013). Moreover, DHEAS exhibited significant positive correlations with BMD at three skeletal sites: including femoral neck, total hip, and lumbar spine (all P < 0.05). Low DHEA and DHEAS levels were associated with increased risk of osteopenia and/or osteoporosis (all P < 0.05) and the risk of osteoporosis gradually decreased with increasing DHEAS levels (P overall = 0.018, P-nonlinear = 0.559). However, DHEA and DHEAS levels in men over the age of 50 with T2DM were not associated with any of above outcomes.
CONCLUSION: In patients with T2DM, independent of testosterone and estradiol, higher DHEA and DHEAS levels are associated with higher BMD and lower risk of osteopenia/osteoporosis in postmenopausal women but not men over the age of 50.

Keywords:  Bone mineral density; Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Osteoporosis; Type 2 diabetes

DOI:  https://doi.org/10.1007/s00774-024-01511-9
Sci Rep. 2024 05 23. 14(1): 11839

Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease.

Kendra L Francis, Hengqi B Zheng, David L Suskind, Taylor A Murphree, Bao Anh Phan, Emily Quah, Aarun S Hendrickson, Xisheng Zhou, Mason Nuding, Alexandra S Hudson, Miklos Guttman, Gregory J Morton, Michael W Schwartz, Kimberly M Alonge, Jarrad M Scarlett.

The intestinal extracellular matrix (ECM) helps maintain appropriate tissue barrier function and regulate host-microbial interactions. Chondroitin sulfate- and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are integral components of the intestinal ECM, and alterations in CS/DS-GAGs have been shown to significantly influence biological functions. Although pathologic ECM remodeling is implicated in inflammatory bowel disease (IBD), it is unknown whether changes in the intestinal CS/DS-GAG composition are also linked to IBD in humans. Our aim was to characterize changes in the intestinal ECM CS/DS-GAG composition in intestinal biopsy samples from patients with IBD using mass spectrometry. We characterized intestinal CS/DS-GAGs in 69 pediatric and young adult patients (n = 13 control, n = 32 active IBD, n = 24 IBD in remission) and 6 adult patients. Here, we report that patients with active IBD exhibit a significant decrease in the relative abundance of CS/DS isomers associated with matrix stability (CS-A and DS) compared to controls, while isomers implicated in matrix instability and inflammation (CS-C and CS-E) were significantly increased. This imbalance of intestinal CS/DS isomers was restored among patients in clinical remission. Moreover, the abundance of pro-stabilizing CS/DS isomers negatively correlated with clinical disease activity scores, whereas both pro-inflammatory CS-C and CS-E content positively correlated with disease activity scores. Thus, pediatric patients with active IBD exhibited increased pro-inflammatory and decreased pro-stabilizing CS/DS isomer composition, and future studies are needed to determine whether changes in the CS/DS-GAG composition play a pathogenic role in IBD.

DOI: https://doi.org/10.1038/s41598-024-60959-x
Int J Biol Macromol. 2024 May 19. pii: S0141-8130(24)03325-7. [Epub ahead of print] 132520

Chondroitin sulfate-based universal nanoparticle delivers angiogenic inhibitor and paclitaxel to exhibit a combination of chemotherapy and anti-angiogenic therapy.

Huiwen Hou, Yan Li, Wen Tang, Didi Gao, Zengmei Liu, Feiyan Zhao, Xinqing Gao, Peixue Ling, Fengshan Wang, Feng Sun, Haining Tan.

  Blocking the tumor nutrient supply through angiogenic inhibitors is an effective treatment approach for malignant tumors. However, using angiogenic inhibitors alone may not be enough to achieve a significant tumor response. Therefore, we recently designed a universal drug delivery system combining chemotherapy and anti-angiogenic therapy to target tumor cells while minimizing drug-related side effects. This system (termed as PCCE) is composed of biomaterial chondroitin sulfate (CS), the anti-angiogenic peptide ES2, and paclitaxel (PTX), which collectively enhance antitumor properties. Interestingly, the PCCE system is conferred exceptional cell membrane permeability due to inherent characteristics of CS, including CD44 receptor-mediated endocytosis. The PCCE could respond to the acidic and high glutathione conditions, thereby releasing PTX and ES2. PCCE could effectively inhibit the proliferation, migration, and invasion of tumor cells and cause apoptosis, while PCCE can affect the endothelial cells tube formation and exert anti-angiogenic function. Consistently, more potent in vivo antitumor efficacy and non-toxic sides were demonstrated in B16F10 xenograft mouse models. PCCE can achieve excellent antitumor activity via modulating angiogenic and apoptosis-related factors. In summary, we have successfully developed an intelligent and responsive CS-based nanocarrier known as PCCE for delivering various antitumor drugs, offering a promising strategy for treating malignant tumors.

Keywords:  Antitumor; Chondroitin sulfate; Drug delivery; Tumor microenvironment; Tumor-targeted

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.132520
Int J Biol Macromol. 2024 May 20. pii: S0141-8130(24)03323-3. [Epub ahead of print] 132518

Identification and characterization of a chondroitinase ABC with a novel carbohydrate-binding module.

Guanchen Liu, Lin Song, Jiajing Li, Xiao Song, Xuanwei Mei, Yuying Zhang, Chuan Fan, Yaoguang Chang, Changhu Xue.

  Chondroitinases play important roles in structural and functional studies of chondroitin sulfates. Carbohydrate-binding module (CBM) is generally considered as an accessory module in carbohydrate-active enzymes, which promotes the association of the appended enzyme with the substrate and potentiates the catalytic activity. However, the role of natural CBM in chondroitinases has not been investigated. Herein, a novel chondroitinase ChABC29So containing an unknown domain with a predicted β-sandwich fold was discovered from Segatella oris. Recombinant ChABC29So showed enzyme activity towards chondroitin sulfates and hyaluronic acid and acted in a random endo-acting manner. The unknown domain exhibited a chondroitin sulfate-binding capacity and was identified as a CBM. Biochemical characterization of ChABC29So and the CBM-truncated enzyme revealed that the CBM enhances the catalytic activity, thermostability, and disaccharide proportion in the final enzymatic products of ChABC29So. These findings first demonstrate the role of the natural CBM in a chondroitinase and will guide future modification of chondroitinases.

Keywords:  Carbohydrate-binding module; Chondroitin sulfate; Chondroitinase ABC

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.132518
bioRxiv. 2024 May 08. pii: 2024.05.07.592968. [Epub ahead of print]

Versican controlled by Lmx1b regulates hyaluronate density and hydration for semicircular canal morphogenesis.

Yusuke Mori, Sierra Smith, Jiacheng Wang, Akankshi Munjal.

  During inner ear semicircular canal morphogenesis in zebrafish, patterned canal-genesis zones express genes for extracellular matrix component synthesis. These include hyaluronan and the hyaluronan-binding chondroitin sulfate proteoglycan Versican, which are abundant in the matrices of many developing organs. Charged hyaluronate polymers play a key role in canal morphogenesis through osmotic swelling. However, the developmental factor(s) that control the synthesis of the matrix components and regulation of hyaluronate density and swelling are unknown. Here, we identify the transcription factor, Lmx1b, as a positive transcriptional regulator of hyaluronan, Versican, and chondroitin synthesis genes crucial for canal morphogenesis. We show that Versican regulates hyaluronan density through its protein core, whereas the charged chondroitin side chains contribute to the osmotic swelling of hyaluronate. Versican-tuned properties of hyaluronate matrices may be a broadly used mechanism in morphogenesis with important implications for understanding diseases where these matrices are impaired, and for hydrogel engineering for tissue regeneration.

Keywords:  Lmx1b; Tissue morphogenesis; Versican; hyaluronan ECM; inner ear semicircular canals; zebrafish

DOI:  https://doi.org/10.1101/2024.05.07.592968
Biomacromolecules. 2024 May 22.

Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.

Lu Fu, Claire A Bridges, Ha Na Kim, Catherine Ding, Nicole Chiwei Bao Hou, Jonathan Yeow, Sandra Fok, Alexander Macmillan, James D Sterling, Shenda M Baker, Megan S Lord.

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

DOI: https://doi.org/10.1021/acs.biomac.4c00477
Glycobiology. 2024 Apr 24. pii: cwae025. [Epub ahead of print]34(6):

Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Marco Maccarana, Binjie Li, Honglian Li, Jianping Fang, Mingjia Yu, Jin-Ping Li.

  Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 μM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Keywords:  DS-epi1; MPS-I; inhibitors; substrate reduction therapy

DOI:  https://doi.org/10.1093/glycob/cwae025
Int J Biol Macromol. 2024 May 16. pii: S0141-8130(24)03244-6. [Epub ahead of print] 132439

Sulfate glycosaminoglycan from swim bladder exerts immunomodulatory potential on macrophages via toll-like receptor 4 mediated NF-κB signaling pathways.

Kun Yang, Xuejing Jia, Jing Chen, Zhuo Wang, Bingbing Song, Rui Li, Kit-Leong Cheong, Saiyi Zhong.

  This study explored the immunomodulatory impact and potential mechanisms on macrophages RAW264.7 using a purified macromolecular sulfate glycosaminoglycan (SBSG) from the swim bladder, whose structure was similar to chondroitin sulfate A. The results showed that SBSG at 0.25-1 mg/mL increased the viability and phagocytosis of RAW264.7 cells. Meanwhile, SBSG promoted the secretion of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and nitric oxide (NO), as well as the production of reactive oxygen species (ROS). According to the RT-PCR and Western blot data, SBSG activated TLR4-nuclear factor kappa B (NF-κB) signaling pathways, which decreased the relative mRNA and protein levels of Toll-like receptor 4 (TLR4), IκB kinase β (IKKβ), NF-κB p65, and p-NF-κB p65. The molecular docking and molecular dynamic simulation findings revealed that the main binding force between TLR4 and SBSG was conventional hydrogen bond interaction, resulting in more stable ligand receptor complexes. In summary, SBSG exhibits significant immunomodulatory potential, similar to chondroitin sulfate C. The underlying molecular mechanism involved the binding of SBSG through hydrogen bonding to TLR4 receptors, triggering the NF-κB signaling pathway to downregulate the expression of related genes and proteins. This, in turn, regulated the secretion of various cytokines that were mediated by macrophages to exert the immunity of the body.

Keywords:  Immunomodulatory mechanism; Macrophages; Molecular docking; Sulfate glycosaminoglycan

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.132439
Int Immunopharmacol. 2024 May 23. pii: S1567-5769(24)00834-8. [Epub ahead of print]135 112314

Renoprotective effect of rosmarinic acid by inhibition of indoxyl sulfate-induced renal interstitial fibrosis via the NLRP3 inflammasome signaling.

Tung-Wei Hung, Yi-Hsien Hsieh, Hsiang-Lin Lee, Yi-Hsuan Ting, Chu-Liang Lin, Wen-Wan Chao.

  We previously reported that rosmarinic acid (RA) ameliorated renal fibrosis in a unilateral ureteral obstruction (UUO) murine model of chronic kidney disease. This study aimed to determine whether RA attenuates indoxyl sulfate (IS)-induced renal fibrosis by regulating the activation of the NLRP3 inflammasome/IL-1β/Smad circuit. We discovered the NLRP3 inflammasome was activated in the IS treatment group and downregulated in the RA-treated group in a dose-dependent manner. Additionally, the downstream effectors of the NLRP3 inflammasome, cleaved-caspase-1 and cleaved-IL-1β showed similar trends in different groups. Moreover, RA administration significantly decreased the ROS levels of reactive oxygen species in IS-treated cells. Our data showed that RA treatment significantly inhibited Smad-2/3 phosphorylation. Notably, the effects of RA on NLRP3 inflammasome/IL-1β/Smad and fibrosis signaling were reversed by the siRNA-mediated knockdown of NLRP3 or caspase-1 in NRK-52E cells. In vivo, we demonstrated that expression levels of NLRP3, c-caspase-1, c-IL-1β, collagen I, fibronectin and α-SMA, and TGF- β 1 were downregulated after treatment of UUO mice with RA or RA + MCC950. Our findings suggested RA and MCC950 synergistically inhibited UUO-induced NLRP3 signaling activation, revealing their renoprotective properties and the potential for combinatory treatment of renal fibrosis and chronic kidney inflammation.

Keywords:  Indoxyl sulfate; MCC950; NLRP3 inflammasome; Rosmarinic acid; Unilateral ureteral obstruction

DOI:  https://doi.org/10.1016/j.intimp.2024.112314
bioRxiv. 2024 May 10. pii: 2024.05.08.593138. [Epub ahead of print]

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing.

Xiuqi Chen, Jasper W van de Sande, Justas Ritmejeris, Chenyu Wen, Henry Brinkerhoff, Andrew H Laszlo, Bauke Albada, Cees Dekker.

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development 1 . Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection 2 . We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level 3 . By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.

DOI: https://doi.org/10.1101/2024.05.08.593138
Gels. 2024 May 14. pii: 330. [Epub ahead of print]10(5):

Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

Paola Bermudez-Lekerika, Katherine B Crump, Karin Wuertz-Kozak, Christine L Le Maitre, Benjamin Gantenbein.

  The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

Keywords:  3D culture; cell viability; intervertebral disc; qPCR; sulfation alginate

DOI:  https://doi.org/10.3390/gels10050330