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.

Sulfotransferase 2B1b, Sterol Sulfonation, and Disease.

The primary function of human sulfotransferase 2B1b (SULT2B1b) is to sulfonate cholesterol and closely related sterols. SULT2B1b sterols perform a number of essential cellular functions. Many are signaling molecules whose activities are redefined by sulfonation-allosteric properties are switched "on" or "off," agonists are transformed into antagonists, and vice versa. Sterol sulfonation is tightly coupled to cholesterol homeostasis, and sulfonation imbalances are causally linked to cholesterol-related diseases including certain cancers, Alzheimer disease, and recessive X-linked ichthyosis-an orphan skin disease. Numerous studies link SULT2B1b activity to disease-relevant molecular processes. Here, these multifaceted processes are integrated into metabolic maps that highlight their interdependence and how their actions are regulated and coordinated by SULT2B1b oxysterol sulfonation. The maps help explain why SULT2B1b inhibition arrests the growth of certain cancers and make the novel prediction that SULT2B1b inhibition will suppress production of amyloid ß (Aß) plaques and tau fibrils while simultaneously stimulating Aß plaque phagocytosis. SULT2B1b harbors a sterol-selective allosteric site whose structure is discussed as a template for creating inhibitors to regulate SULT2B1b and its associated biology. SIGNIFICANCE STATEMENT: Human sulfotransferase 2B1b (SULT2B1b) produces sterol-sulfate signaling molecules that maintain the homeostasis of otherwise pro-disease processes in cancer, Alzheimer disease, and X-linked ichthyosis-an orphan skin disease. The functions of sterol sulfates in each disease are considered and codified into metabolic maps that explain the interdependencies of the sterol-regulated networks and their coordinate regulation by SULT2B1b. The structure of the SULT2B1b sterol-sensing allosteric site is discussed as a means of controlling sterol sulfate biology.

NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.

Lysosomes are key organelles maintaining cellular homeostasis in health and disease. Here, we report the identification of N-deacetylase and N-sulfotransferase 3 (NDST3) as a potent regulator of lysosomal functions through an unbiased genetic screen. NDST3 constitutes a new member of the histone deacetylase (HDAC) family and catalyzes the deacetylation of α-tubulin. Loss of NDST3 promotes assembly of the V-ATPase holoenzyme on the lysosomal membrane and thereby increases the acidification of the organelle. NDST3 is downregulated in tissues and cells from patients carrying the C9orf72 hexanucleotide repeat expansion linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Deficiency in C9orf72 decreases the level of NDST3, and downregulation of NDST3 exacerbates the proteotoxicity of poly-dipeptides generated from the C9orf72 hexanucleotide repeats. These results demonstrate a previously unknown regulatory mechanism through which microtubule acetylation regulates lysosomal activities and suggest that NDST3 could be targeted to modulate microtubule and lysosomal functions in relevant diseases.

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

Prevention of experimental autoimmune encephalomyelitis by targeting 6-sulfo sialyl Lewis X glycans involved in lymphocyte homing.

Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.

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.

Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.

HYPOTHESIS: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration. METHODS AND RESULTS: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS. CONCLUSION: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

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.

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.

SULF1 expression is increased and promotes fibrosis through the TGF-ß1/SMAD pathway in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology. Despite the increasing global incidence and poor prognosis, the exact pathogenic mechanisms remain elusive. Currently, effective therapeutic targets and treatment methods for this disease are still lacking. This study tried to explore the pathogenic mechanisms of IPF. We found elevated expression of SULF1 in lung tissues of IPF patients compared to normal control lung tissues. SULF1 is an enzyme that modifies heparan sulfate chains of heparan sulfate proteoglycans, playing a critical role in biological regulation. However, the effect of SULF1 in pulmonary fibrosis remains incompletely understood. Our study aimed to investigate the impact and mechanisms of SULF1 in fibrosis. METHODS: We collected lung specimens from IPF patients for transcriptome sequencing. Validation of SULF1 expression in IPF patients was performed using Western blotting and RT-qPCR on lung tissues. ELISA experiments were employed to detect SULF1 concentrations in IPF patient plasma and TGF-ß1 levels in cell culture supernatants. We used lentiviral delivery of SULF1 shRNA to knock down SULF1 in HFL1 cells, evaluating its effects on fibroblast secretion, activation, proliferation, migration, and invasion capabilities. Furthermore, we employed Co-Immunoprecipitation (Co-IP) to investigate the regulatory mechanisms involved. RESULTS: Through bioinformatic analysis of IPF transcriptomic sequencing data (HTIPF) and datasets GSE24206, and GSE53845, we identified SULF1 may potentially play a crucial role in IPF. Subsequently, we verified that SULF1 was upregulated in IPF and predominantly increased in fibroblasts. Furthermore, SULF1 expression was induced in HFL1 cells following exposure to TGF-ß1. Knockdown of SULF1 suppressed fibroblast secretion, activation, proliferation, migration, and invasion under both TGF-ß1-driven and non-TGF-ß1-driven conditions. We found that SULF1 catalyzes the release of TGF-ß1 bound to TGFßRIII, thereby activating the TGF-ß1/SMAD pathway to promote fibrosis. Additionally, TGF-ß1 induces SULF1 expression through the TGF-ß1/SMAD pathway, suggesting a potential positive feedback loop between SULF1 and the TGF-ß1/SMAD pathway. CONCLUSIONS: Our findings reveal that SULF1 promotes fibrosis through the TGF-ß1/SMAD pathway in pulmonary fibrosis. Targeting SULF1 may offer a promising therapeutic strategy against IPF.

SULT2B1-CS-DOCK2 axis regulates effector T-cell exhaustion in HCC microenvironment.

BACKGROUND AND AIMS: HCC is a malignant disease. Compared with tyrosine kinase inhibitors (the classical therapy), immune checkpoint inhibitors are more effective in the treatment of HCC, despite their limited efficacy. Among these restricted factors, exhaustion of tumor-infiltrated lymphocytes, especially CD8 + T cells, is a core event. We aimed to determine the key factors contributing to CD8 + T-cell infiltration in HCC and investigate the underlying mechanisms. APPROACH AND RESULTS: Using machine learning and multiplex immunohistochemistry analysis, we showed that dedicator of cytokinesis protein 2 (DOCK2) was a potential indicator of infiltrated CD8 + T cells in HCC. Using RNA sequencing, flow cytometry analysis, and mouse HCC models, we demonstrated that DOCK2 inactivation accounted for infiltrated CD8 + T-cell exhaustion in tumors. Using quasi-targeted metabolomics, mass spectrum, and mass cytometry by time of flight analysis, we found that cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells suppressed DOCK2 enzymatic activity of T cells. Through virtual screening, molecular docking simulation, and experiments validation, we demonstrated that tolazamide reversed DOCK2 inactivation-mediated CD8 + T-cell exhaustion and enhanced anti-programmed death-ligand 1 antibody+apatinib immunotherapeutic effects on HCC. CONCLUSIONS: This study indicates that DOCK2 controls CD8 + T-cell infiltration in HCC, and cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells promotes effector T-cell exhaustion. The findings suggest that the usage of conventional drugs affects immunotherapy efficacy in HCC patients.

Dissection of N-deacetylase and N-sulfotransferase activities of NDST1 and their effects on Wnt8 distribution and signaling in Xenopus embryos.

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero-posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N-deacetylase/N-sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N-sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N-sulfation, of HS chains in Xenopus embryos. Unlike wild-type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP-Flash reporter assay. These results suggest that N-sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild-type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1.

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.

Identification and characterization of sulphotransferase (SOT) genes for tolerance against drought and heat in wheat and six related species.

BACKGROUND: Sulphotransferase (SOT) enzyme (encoded by a conserved family of SOT genes) is involved in sulphonation of a variety of compounds, through transfer of a sulphuryl moiety from 3'phosphoadenosine- 5'phosphosulphate (PAPS) to a variety of secondary metabolites. The PAPS itself is derived from 3'adenosine-5'phosphosulphate (APS) that is formed after uptake of sulphate ions from the soil. The process provides tolerance against abiotic stresses like drought and heat in plants. Therefore, a knowledge of SOT genes in any crop may help in designing molecular breeding methods for improvement of tolerance for drought and heat. METHODS: Sequences of rice SOT genes and SOT domain (PF00685) of corresponding proteins were both used for identification of SOT genes in wheat and six related species (T. urartu, Ae. tauschii, T. turgidum, Z. mays, B. distachyon and Hordeum vulgare), although detailed analysis was conducted only in wheat. The wheat genes were mapped on individual chromosomes and also subjected to synteny and collinearity analysis. The proteins encoded by these genes were examined for the presence of a complete SOT domain using 'Conserved Domain Database' (CDD) search tool at NCBI. RESULTS: In wheat, 107 TaSOT genes, ranging in length from 969 bp to 7636 bp, were identified and mapped onto individual chromosomes. SSRs (simple sequence repeats), microRNAs, long non-coding RNAs (lncRNAs) and their target sites were also identified in wheat SOT genes. SOT proteins were also studied in detail. An expression assay of TaSOT genes via wheat RNA-seq data suggested engagement of these genes in growth, development and responses to various hormones and biotic/abiotic stresses. CONCLUSIONS: The results of the present study should help in further functional characterization of SOT genes in wheat and other related crops.

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.

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.

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes.

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19) to the extent of 16%, together catalyzing 43% of the reactions, followed by P450 Subfamily 1A (P450 1A1 and P450 1A2) with 15%. The P450 2D6 enzyme participated in an extent of 8%, P450 2E1 in 10%, and P450 2B6 in 6% of the reactions. All other enzymes participate to the extent of 14%. The data show that, of the human enzymes analyzed, P450 enzymes were dominant in catalyzing potential toxication reactions of drugs and their metabolites, with the major role assigned to the P450 Subfamily 3A and significant participation of the P450 Subfamilies 2C and 1A, plus the 2D6, 2E1 and 2B6 enzymes contributing. Selected examples of drugs that are activated or proposed to form toxic species are discussed.

Tubulin deacetylase NDST3 modulates lysosomal acidification: Implications in neurological diseases.

Neurological diseases (NDs), featured by progressive dysfunctions of the nervous system, have become a growing burden for the aging populations. N-Deacetylase and N-sulfotransferase 3 (NDST3) is known to catalyze deacetylation and N-sulfation on disaccharide substrates. Recently, NDST3 is identified as a novel deacetylase for tubulin, and its newly recognized role in modulating microtubule acetylation and lysosomal acidification provides fresh insights into ND therapeutic approaches using NDST3 as a target. Microtubule acetylation and lysosomal acidification have been reported to be critical for activities in neurons, implying that the regulators of these two biological processes, such as the previously known microtubule deacetylases, histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), could play important roles in various NDs. Aberrant NDST3 expression or tubulin acetylation has been observed in an increasing number of NDs, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), schizophrenia and bipolar disorder, Alzheimer's disease (AD), and Parkinson's disease (PD), suggesting that NDST3 is a key player in the pathogenesis of NDs and may serve as a target for development of new treatment of NDs.

Hepatic enzyme induction and its potential effect on thyroid hormone metabolism in the metamorphosing tadpole of Xenopus laevis (African clawed frog).

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-ß-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.