The extracellular heparan sulfatase SULF2 limits myeloid IFNß signaling and Th17 responses in inflammatory arthritis.

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/- bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/- chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology.

Genome-wide CRISPR screening identifies tyrosylprotein sulfotransferase-2 as a target for augmenting anti-PD1 efficacy.

BACKGROUND: Immune checkpoint therapy (ICT) provides durable responses in select cancer patients, yet resistance remains a significant challenge, prompting the exploration of underlying molecular mechanisms. Tyrosylprotein sulfotransferase-2 (TPST2), known for its role in protein tyrosine O-sulfation, has been suggested to modulate the extracellular protein-protein interactions, but its specific role in cancer immunity remains largely unexplored. METHODS: To explore tumor cell-intrinsic factors influencing anti-PD1 responsiveness, we conducted a pooled loss-of-function genetic screen in humanized mice engrafted with human immune cells. The responsiveness of cancer cells to interferon-γ (IFNγ) was estimated by evaluating IFNγ-mediated induction of target genes, STAT1 phosphorylation, HLA expression, and cell growth suppression. The sulfotyrosine-modified target gene of TPST2 was identified by co-immunoprecipitation and mass spectrometry. The in vivo effects of TPST2 inhibition were evaluated using mouse syngeneic tumor models and corroborated by bulk and single-cell RNA sequencing analyses. RESULTS: Through in vivo genome-wide CRISPR screening, TPST2 loss-of-function emerged as a potential enhancer of anti-PD1 treatment efficacy. TPST2 suppressed IFNγ signaling by sulfating IFNγ receptor 1 at Y397 residue, while its downregulation boosted IFNγ-mediated signaling and antigen presentation. Depletion of TPST2 in cancer cells augmented anti-PD1 antibody efficacy in syngeneic mouse tumor models by enhancing tumor-infiltrating lymphocytes. RNA sequencing data revealed TPST2's inverse correlation with antigen presentation, and increased TPST2 expression is associated with poor prognosis and altered cancer immunity across cancer types. CONCLUSIONS: We propose TPST2's novel role as a suppressor of cancer immunity and advocate for its consideration as a therapeutic target in ICT-based treatments.

Biosynthesis of animal-free recombinant chondroitin sulfate E using a functional chondroitin sulfotransferase in E. coli.

Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli. Moreover, there is no experimentally derived three-dimensional structure available for this specific sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks. KEY POINTS: • Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished. • First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.

Sulfotransferase 4A1 Coding Sequence and Protein Structure Are Highly Conserved in Vertebrates.

Cytosolic sulfotransferases (SULTs) are Phase 2 drug-metabolizing enzymes that catalyze the conjugation of sulfonate to endogenous and xenobiotic compounds, increasing their hydrophilicity and excretion from cells. To date, 13 human SULTs have been identified and classified into five families. SULT4A1 mRNA encodes two variants: (1) the wild type, encoding a 284 amino acid, ~33 kDa protein, and (2) an alternative spliced variant resulting from a 126 bp insert between exon 6 and 7, which introduces a premature stop codon that enhances nonsense-mediated decay. SULT4A1 is classified as an SULT based on sequence and structural similarities, including PAPS-domains, active-site His, and the dimerization domain; however, the catalytic pocket lid 'Loop 3' size is not conserved. SULT4A1 is uniquely expressed in the brain and localized in the cytosol and mitochondria. SULT4A1 is highly conserved, with rare intronic polymorphisms that have no outward manifestations. However, the SULT4A1 haplotype is correlated with Phelan-McDermid syndrome and schizophrenia. SULT4A1 knockdown revealed potential SULT4A1 functions in photoreceptor signaling and knockout mice display hampered neuronal development and behavior. Mouse and yeast models revealed that SULT4A1 protects the mitochondria from endogenously and exogenously induced oxidative stress and stimulates cell division, promoting dendritic spines' formation and synaptic transmission. To date, no physiological enzymatic activity has been associated with SULT4A1.

GAL3ST1 Deficiency Reduces Epithelial-Mesenchymal Transition and Tumorigenic Capacity in a Cholangiocarcinoma Cell Line.

Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1-deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

A Novel Fluorescence-Based Microplate Assay for High-Throughput Screening of hSULT1As Inhibitors.

Human sulfotransferase 1As (hSULT1As) play a crucial role in the metabolic clearance and detoxification of a diverse range of endogenous and exogenous substances, as well as in the bioactivation of some procarcinogens and promutagens. Pharmacological inhibiting hSULT1As activities may enhance the in vivo effects of most hSULT1As drug substrates and offer protective strategies against the hSULT1As-mediated bioactivation of procarcinogens. To date, a fluorescence-based high-throughput assay for the efficient screening of hSULT1As inhibitors has not yet been reported. In this work, a fluorogenic substrate (HN-241) for hSULT1As was developed through scaffold-seeking and structure-guided molecular optimization. Under physiological conditions, HN-241 could be readily sulfated by hSULT1As to form HN-241 sulfate, which emitted brightly fluorescent signals around 450 nm. HN-241 was then used for establishing a novel fluorescence-based microplate assay, which strongly facilitated the high-throughput screening of hSULT1As inhibitors. Following the screening of an in-house natural product library, several polyphenolic compounds were identified with anti-hSULT1As activity, while pectolinarigenin and hinokiflavone were identified as potent inhibitors against three hSULT1A isozymes. Collectively, a novel fluorescence-based microplate assay was developed for the high-throughput screening and characterization of hSULT1As inhibitors, which offered an efficient and facile approach for identifying potent hSULT1As inhibitors from compound libraries.

Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Knockdown of sulfotransferase 2B1 suppresses cell migration, invasion and promotes apoptosis in ovarian carcinoma cells via targeting annexin A9.

BACKGROUND: Sulfotransferase family 2B member 1 (SULT2B1) has been reported to play oncogenic role in many types of cancers. Nevertheless, the role that SULT2B1 played in ovarian cancer (OC) and the hidden molecular mechanism is obscure. METHODS: Expression of SULT2B1 in OC was analyzed by GEPIA database. qRT-PCR and western blot (WB) was applied for the appraisement of SULT2B1 and Annexin A9 (ANXA9) in OC cell lines. The capabilities of cells to proliferate, migrate and invade were assessed with CCK-8 assay, wound healing assay, along with transwell assay. Cell apoptotic level was estimated utilizing flow cytometry. WB was employed for the evaluation of migration- and apoptosis-related proteins. Bioinformatic analysis and co-immunoprecipitation were used to predict and verify the combination of SULT2B1 and ANXA9. RESULTS: The data showed that SULT2B1 and ANXA9 were upregulated in OC cells. SULT2B1 depletion suppressed the proliferative, migrative, and invasive capabilities of SKOV3 cells but facilitated the cell apoptosis. SULT2B1-regulated ANXA9 expression and were proved to bind to ANXA9. Additionally, ANXA9 deficiency exhibited the same impacts on cell migrative, invasive capability and apoptotic level as SULT2B1 silencing. Moreover, ANXA9 overexpression reversed the inhibitory impacts of SULT2B1 silencing on the proliferative, migrative, invasive, and apoptotic capabilities of SKOV3 cells. CONCLUSION: In summary, SULT2B1 silencing repressed OC progression by targeting ANXA9.

In silico Screening of Food and Drug Administration-approved Compounds against Trehalose 2-sulfotransferase (Rv0295c) in Mycobacterium tuberculosis: Insights from Molecular Docking and Dynamics Simulations.

BACKGROUND: Tuberculosis (TB) remains a prominent global health challenge, distinguished by substantial occurrences of infection and death. The upsurge of drug-resistant TB strains underscores the urgency to identify novel therapeutic targets and repurpose existing compounds. Rv0295c is a potentially druggable enzyme involved in cell wall biosynthesis and virulence. We evaluated the inhibitory activity of Food and Drug Administration (FDA)-approved compounds against Rv0295c of Mycobacterium tuberculosis, employing molecular docking, ADME evaluation, and dynamics simulations. METHODS: The study screened 1800 FDA-approved compounds and selected the top five compounds with the highest docking scores. Following this, we subjected the initially screened ligands to ADME analysis based on their dock scores. In addition, the compound exhibited the highest binding affinity chosen for molecular dynamics (MD) simulation to investigate the dynamic behavior of the ligand-receptor complex. RESULTS: Dihydroergotamine (CHEMBL1732) exhibited the highest binding affinity (-12.8 kcal/mol) for Rv0295c within this set of compounds. We evaluated the stability and binding modes of the complex over extended simulation trajectories. CONCLUSION: Our in silico analysis demonstrates that FDA-approved drugs can serve as potential Rv0295c inhibitors through repurposing. The combination of molecular docking and MD simulation offers a comprehensive understanding of the interactions between ligands and the protein target, providing valuable guidance for further experimental validation. Identifying Rv0295c inhibitors may contribute to new anti-TB drugs.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Nanoparticle/Engineered Bacteria Based Triple-Strategy Delivery System for Enhanced Hepatocellular Carcinoma Cancer Therapy.

Background: New treatment modalities for hepatocellular carcinoma (HCC) are desperately critically needed, given the lack of specificity, severe side effects, and drug resistance with single chemotherapy. Engineered bacteria can target and accumulate in tumor tissues, induce an immune response, and act as drug delivery vehicles. However, conventional bacterial therapy has limitations, such as drug loading capacity and difficult cargo release, resulting in inadequate therapeutic outcomes. Synthetic biotechnology can enhance the precision and efficacy of bacteria-based delivery systems. This enables the selective release of therapeutic payloads in vivo. Methods: In this study, we constructed a non-pathogenic Escherichia coli (E. coli) with a synchronized lysis circuit as both a drug/gene delivery vehicle and an in-situ (hepatitis B surface antigen) Ag (ASEc) producer. Polyethylene glycol (CHO-PEG2000-CHO)-poly(ethyleneimine) (PEI25k)-citraconic anhydride (CA)-doxorubicin (DOX) nanoparticles loaded with plasmid encoded human sulfatase 1 (hsulf-1) enzyme (PNPs) were anchored on the surface of ASEc (ASEc@PNPs). The composites were synthesized and characterized. The in vitro and in vivo anti-tumor effect of ASEc@PNPs was tested in HepG2 cell lines and a mouse subcutaneous tumor model. Results: The results demonstrated that upon intravenous injection into tumor-bearing mice, ASEc can actively target and colonise tumor sites. The lytic genes to achieve blast and concentrated release of Ag significantly increased cytokine secretion and the intratumoral infiltration of CD4/CD8+T cells, initiated a specific immune response. Simultaneously, the PNPs system releases hsulf-1 and DOX into the tumor cell resulting in rapid tumor regression and metastasis prevention. Conclusion: The novel drug delivery system significantly suppressed HCC in vivo with reduced side effects, indicating a potential strategy for clinical HCC therapy.

Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Macular corneal dystrophy with iridofundal coloboma in the same patient: a unique combination.

A young a presented with painless, progressive diminution of vision in both eyes (BE). Slit lamp examination revealed the presence of a single central corneal opacity in the right eye and multiple corneal opacities of varying sizes in the left eye (LE), limited to the anterior-mid corneal stroma. Microcornea with reduced central corneal thickness and complete inferonasal iris coloboma along with inferior fundal coloboma, sparing both the disc and macula, were noted in BE. A diagnosis of BE macular corneal dystrophy (MCD) and iridofundal coloboma (IFC) was made. The patient underwent LE sutureless anterior lamellar therapeutic keratoplasty. On histopathological examination, the excised corneal tissue revealed stromal lamellar disarray with positive colloidal iron staining, strongly suggestive of MCD. Whole-exome sequencing revealed the presence of a likely pathogenic carbohydrate sulfotransferase 6 (CHST6) mutation, confirming the diagnosis of MCD. This concurrent presence of IFC with a corneal stromal dystrophy is previously unreported in the literature, to the best of our knowledge.

Exploring the impact of insufficient thermal ablation on hepatocellular carcinoma: NDST2 overexpression mechanism and its role in facilitating growth and invasion of residual cancer cells.

OBJECTIVE: Incomplete thermal ablation (ITA) fosters the malignancy of residual cells in Hepatocellular carcinoma (HCC) with unclear mechanisms now. This study aims to investigate the expression changes of NDST2 following ITA of HCC and its impact on residual cancer cells. METHODS: An in vitro model of heat stress-induced liver cancer was constructed to measure the expression of NDST2 using Quantitative Real-Time PCR and Western blotting experiments. The sequencing data from nude mice were used for validation. The clinical significance of NDST2 in HCC was evaluated by integrating datasets. Gene ontology and pathway analysis were conducted to explore the potential signaling pathways regulated by NDST2. Additionally, NDST2 was knocked down in heat stress-induced HCC cells, and the effects of NDST2 on these cells were verified using Cell Counting Kit-8 assays, scratch assays, and Transwell assays. RESULTS: NDST2 expression levels are elevated in HCC, leading to a decrease in overall survival rates of HCC patients. Upregulation of immune checkpoint levels in high NDST2-expressing HCC may contribute to immune evasion by liver cancer cells. Additionally, the low mutation rate of NDST2 in HCC suggests a relatively stable expression of NDST2 in this disease. Importantly, animal and cell models treated with ITA demonstrate upregulated expression of NDST2. Knockdown of NDST2 in heat stress-induced liver cancer cells results in growth inhibition associated with gene downregulation. CONCLUSION: The upregulation of NDST2 can accelerate the progression of residual HCC after ITA, suggesting a potential role for NDST2 in the therapeutic efficacy and prognosis of residual HCC.

Syndecans, Exostosins and Sulfotransferases as Potential Synovial Inflammation Moderators in Patients with Hip Osteoarthritis.

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.

Crystal structure of activating sulfotransferase SgdX2 involved in biosynthesis of secondary metabolite sungeidine.

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3'-phosphoadenosine 5'-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

Effect of CHST11, a novel biomarker, on the biological functionalities of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor, and the role of carbohydrate sulfotransferase 11 (CHST11) in this cancer remains unclear. Here, by using bioinformatics methods, we comprehensively analyzed the relationship between CHST11 and clinical significance, immune infiltration, functional enrichment, m6A methylation, and protein-protein interaction networks. We found that CHST11 expression was significantly higher in ccRCC samples than in normal tissues. Additionally, CHST11 levels correlated with the clinicopathological features of ccRCC patients and functioned as a prognostic factor for patient survival. Functional analysis revealed the involvement of CHST11 in metabolic pathways. Immune infiltration and m6A methylation analysis suggested the association of CHST11 with immune cell abundance in the tumor microenvironment and specific methylation patterns in ccRCC. The in vitro analysis of the clinical samples and ccRCC cell lines demonstrated that the overexpression of CHST11 promotes ccRCC cell proliferation, migration, and invasion, while its suppression has the opposite effect. Thus, CHST11 may play a remarkable role in the occurrence and progression of ccRCC. Functionally, CHST11 promotes the aggressiveness of ccRCC cells. These findings provide insights into the role of CHST11 in ccRCC progression.Registry and the Registration No. of the study/trial: No. 2021K034.