bims-supasi Biomed News
on Sulfation pathways and signalling
Issue of 2024‒06‒23
nine papers selected by
Jonathan Wolf Mueller, University of Birmingham

  1. Mol Cell Proteomics. 2024 Jun 14. pii: S1535-9476(24)00093-8. [Epub ahead of print] 100803
      Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus (LH) and the striatum (ST). We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the LH by adeno-associated virus (AAV) delivery of an shRNA to Arylsulfatase B (N-acetylgalactosamine-4-sulfatase, ARSB) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated vs. saline-treated mice, including MYPR, KCC2A, SYN2, TENR, CALX, ANXA7, HDGF, NCAN, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.
    Keywords:  Addiction; chondroitin sulfate; cocaine; drug abuse; glycomics; glycosaminoglycans; heparan sulfate; lateral hypothalamus; mass-spectrometry; methamphetamine; neurobiology; proteoglycans; proteomics; striatum
  2. Int J Mol Sci. 2024 May 28. pii: 5897. [Epub ahead of print]25(11):
      Oil-core nanocapsules (NCs, also known as nanoemulsions) are of great interest due to their application as efficient carriers of various lipophilic bioactives, such as drugs. Here, we reported for the first time the preparation and characterization of NCs consisting of chondroitin sulfate (CS)-based shells and liquid oil cores. For this purpose, two amphiphilic CS derivatives (AmCSs) were obtained by grafting the polysaccharide chain with octadecyl or oleyl groups. AmCS-based NCs were prepared by an ultrasound-assisted emulsification of an oil phase consisting of a mixture of triglyceride oil and vitamin E in a dispersion of AmCSs. Dynamic light scattering and cryo-transmission electron microscopy showed that the as-prepared core-shell NCs have typical diameters in the range of 30-250 nm and spherical morphology. Since CS is a strong polyanion, these particles have a very low surface potential, which promotes their stabilization. The cytotoxicity of the CS derivatives and CS-based NCs and their impact on cell proliferation were analyzed using human keratinocytes (HaCaTs) and primary human skin fibroblasts (HSFs). In vitro studies showed that AmCSs dispersed in an aqueous medium, exhibiting mild cytotoxicity against HaCaTs, while for HSFs, the harmful effect was observed only for the CS derivative with octadecyl side groups. However, the nanocapsules coated with AmCSs, especially those filled with vitamin E, show high biocompatibility with human skin cells. Due to their stability under physiological conditions, the high encapsulation efficiency of their hydrophobic compounds, and biocompatibility, AmCS-based NCs are promising carriers for the topical delivery of lipophilic bioactive compounds.
    Keywords:  bioactives; chondroitin sulfate; cryo-transmission electron microscopy; cytotoxicity; nanocapsules
  3. J Proteome Res. 2024 Jun 20.
      Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.
    Keywords:  cysteinyl-glycine; direct PTM determination; electron capture dissociation; electron transfer dissociation; post-translational modifications; proteomics; site localization, mass spectrometry; tyrosine sulfation
  4. Int J Mol Sci. 2024 Jun 06. pii: 6281. [Epub ahead of print]25(11):
      Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.
    Keywords:  endocytosis; heparan sulfate degradation; heparanase; heparastatin (SF4); heparin; mast cells; syndecan
  5. Int J Mol Sci. 2024 May 28. pii: 5851. [Epub ahead of print]25(11):
      The effects of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate, on the expression of PD-L1 were determined, and the underlying mechanism of PD-L1 expression was elucidated. Initial experiments in human melanoma cells (A375) showed that PD-L1 expression increased from 357 ± 31 to 796 ± 50 pg/mg protein (p < 10-11) when ARSB was silenced in A375 cells. In subcutaneous B16F10 murine melanomas, PD-L1 declined from 1227 ± 189 to 583 ± 110 pg/mg protein (p = 1.67 × 10-7), a decline of 52%, following treatment with exogenous, bioactive recombinant ARSB. This decline occurred in association with reduced tumor growth and prolongation of survival, as previously reported. The mechanism of regulation of PD-L1 expression by ARSB is attributed to ARSB-mediated alteration in chondroitin 4-sulfation, leading to changes in free galectin-3, c-Jun nuclear localization, HDAC3 expression, and effects of acetyl-H3 on the PD-L1 promoter. These findings indicate that changes in ARSB contribute to the expression of PD-L1 in melanoma and can thereby affect the immune checkpoint response. Exogenous ARSB acted on melanoma cells and normal melanocytes through the IGF2 receptor. The decline in PD-L1 expression by exogenous ARSB may contribute to the impact of ARSB on melanoma progression.
    Keywords:  HDAC3; arylsulfatase B; c-Jun; chondroitin sulfate; galectin-3
  6. Mol Neurobiol. 2024 Jun 20.
      Chondroitin sulfate proteoglycans (CSPGs) and proteoglycan receptor protein tyrosine phosphatase σ (PTPσ) play a critical role in the pathology of spinal cord injury (SCI). CSPGs can be induced by autophagy inhibition in astrocyte. However, CSPG's impact on autophagy and its role in SCI is still unknown. We investigate intracellular sigma peptide (ISP) targeting PTPσ, its effects on autophagy, and synaptic reorganization in SCI. We found that ISP increased the level of autophagosome marker LC3B-II/I and decreased autophagosome degradation marker p62 in SCI, suggesting activated autophagy flux. ISP restored autophagosome-lysosome fusion-related protein syntaxin 17 (STX17) and lysosome-associated membrane protein 2 (LAMP2), indicating activated autophagosome-lysosome fusion. ISP increased pre-synaptic marker synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) expression and improved excitatory synapse marker vesicular glutamate transporter 1 (VGLUT1) and SYN in SCI, suggesting improved synaptic reorganization. ISP promoted axon marker neurofilament and growth-related GAP-43 expression in SCI. ISP rescued a preserved number of motor neurons and improved neurobehavioral recovery after SCI. Our study extended the CSPG-PTPσ inhibition role in activating autophagy flux, axon and synaptic reorganization, and functional recovery in SCI.
    Keywords:  Autophagy flux; CSPG; Lysosome fusion; PTPσ; Spinal cord injury
  7. bioRxiv. 2024 Jun 06. pii: 2024.06.04.597413. [Epub ahead of print]
      Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophage's ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma.STATEMENT OF SIGNIFICANCE: We engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth in vivo . Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.
  8. Kidney Int Rep. 2024 Jun;9(6): 1674-1683
      Introduction: Growth failure is considered the most important clinical outcome parameter in childhood chronic kidney disease (CKD). Central to the pathophysiology of growth failure is the presence of a chronic proinflammatory state, presumed to be partly driven by the accumulation of uremic toxins. In this study, we assessed the association between uremic toxin concentrations and height velocity in a longitudinal multicentric prospective pediatric CKD cohort of (pre)school-aged children and children during pubertal stages.Methods: In a prospective, multicentric observational study, a selection of uremic toxin levels of children (aged 0-18 years) with CKD stage 1 to 5D was assessed every 3 months (maximum 2 years) along with clinical growth parameters. Linear mixed models with a random slope for age and a random intercept for child were fitted for height (in cm and SD scores [SDS]). A piecewise linear association between age and height was assumed.
    Results: Data analysis included data from 560 visits of 81 children (median age 9.4 years; 2/3 male). In (pre)school aged children (aged 2-12 years), a 10% increase in concurrent indoxyl sulfate (IxS, total) concentration resulted in an estimated mean height velocity decrease of 0.002 SDS/yr (P < 0.05), given that CKD stage, growth hormone (GH), bicarbonate concentration, and dietary protein intake were held constant. No significant association with height velocity was found in children during pubertal stages (aged >12 years).
    Conclusion: The present study demonstrated that, especially IxS contributes to a lower height velocity in (pre)school children, whereas we could not find a role for uremic toxins with height velocity during pubertal stages.
    Keywords:  child; chronic kidney disease; dialysis; growth failure; inflammation; uremic toxins
  9. J Gen Appl Microbiol. 2024 Jun 18.
      In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.
    Keywords:  Site-directed mutagenesis; Substrate specificity; Sulfotransferase assay; Yeast 3’,5’-bisphosphate nucleotidase