bims-supasi 2023-08-20 papers

Biosens Bioelectron. 2023 Aug 12. pii: S0956-5663(23)00537-7. [Epub ahead of print]238 115595

Development of peptide-based ratiometric fluorescent probe for sensing heparan sulfate and heparin in aqueous solutions at physiological pH and quantitative detection of heparan sulfate in live cells.

Sumita Subedi, Hyun Jung Hwang, Donghee Kang, Pramod Kumar Mehta, Nayeon Kim, Hyojin Park, Jae-Seon Lee, Keun-Hyeung Lee.

Heparan sulfate (HS) plays a critical role in various biological processes as a vital component of the extracellular matrix. In this study, we synthesized three fluorescent probes (1-3) comprising Arg-rich peptides as HS receptors and a fluorophore capable of exhibiting red-shifted emissions upon aggregation. All three probes demonstrated ratiometric responses to HS and heparin in aqueous solutions. Remarkably, probe 3 exhibited a unique ratiometric response to HS in both aqueous solutions at physiological pH and HS proteoglycans on live cells. Probe 3 displayed exceptional sensing properties, including high biocompatibility, water solubility, visible light excitation, a large Stokes shift for ratiometric detection and remarkable selectivity and sensitivity for HS (with a low limit of detection: 720 pM). Binding mode studies unveiled the crucial role of charge interactions between probe 3 and negatively charged HS sugar units. Upon binding, the fluorophore segments of the probes overlapped, inducing green and red emission changes through restricted intramolecular rotation of the fluorophore moiety. Importantly, probe 3 was effectively employed to quantify the reduction of HS proteoglycan levels in live cells by inhibiting HS sulfation using siRNA and an inhibitor. It successfully detected decreased HS levels in cells treated with doxorubicin and irradiation, consistent with results obtained from western blot and immunofluorescence assays. This study presents the first ratiometric fluorescent probe capable of quantitatively detecting HS levels in aqueous solutions and live cells. The unique properties of peptide-based probe 3 make it a valuable tool for studying HS biology and potentially for diagnostic applications in various biological systems.

DOI: https://doi.org/10.1016/j.bios.2023.115595

Biochem Biophys Res Commun. 2023 Aug 11. pii: S0006-291X(23)00958-0. [Epub ahead of print]677 149-154

Crystal structure of Arabidopsis thaliana sulfotransferase SOT16 involved in glucosinolate biosynthesis.

Yuka Iwamoto, Seira Saito, Takamasa Teramoto, Akiko Maruyama-Nakashita, Yoshimitsu Kakuta.

Glucosinolates (GSLs), a class of secondary metabolites found in Brassicaceae plants, play important roles in plant defense and contribute distinct flavors and aromas when used as food ingredients. Following tissue damage, GSLs undergo enzymatic hydrolysis to release bioactive volatile compounds. Understanding GSL biosynthesis and enzyme involvement is crucial for improving crop quality and advancing agriculture. Plant sulfotransferases (SOTs) play a key role in the final step of GSL biosynthesis by transferring sulfate groups to the precursor molecules. In the present study, we investigated the enzymatic reaction mechanism and broad substrate specificity of Arabidopsis thaliana sulfotransferase AtSOT16, which is involved in GSL biosynthesis, using crystal structure analysis. Our analysis revealed the specific catalytic residues involved in the sulfate transfer reaction and supported the hypothesis of a concerted acid-base catalytic mechanism. Furthermore, the docking models showed a strong correlation between the substrates with high predicted binding affinities and those experimentally reported to exhibit high activity. These findings provide valuable insights into the enzymatic reaction mechanisms and substrate specificity of GSL biosynthesis. The information obtained in this study may contribute to the development of novel strategies for manipulating GSL synthesis pathways in Brassica plants and has potential agricultural applications.

Keywords: Cytosolic sulfotransferase; Enzymology; Glucosinolate biosynthesis; Sulfation; X-ray crystallography

DOI: https://doi.org/10.1016/j.bbrc.2023.08.020

Ther Apher Dial. 2023 Aug 19.

Effects of N-acetyl-L-tryptophan on desorption of the protein-bound uremic toxin indoxyl sulfate and effects on uremic sarcopenia.

Atsushi Ohashi, Masashi Nakatani, Hideo Hori, Shigeru Nakai, Kunihiro Tsuchida, Midori Hasegawa, Naotake Tsuboi.

INTRODUCTION: Indoxyl sulfate (IS) is a protein-bound uremic toxin that causes uremic sarcopenia. IS has poor dialysis clearance; however, the addition of a binding competitor improves its removal efficiency.
METHODS: Dialysis experiments were performed using N-acetyl-l-tryptophan (L-NAT) instead of l-tryptophan (Trp) using pooled sera obtained from dialysis patients. The molecular structures of L-NAT and Trp were similar to that of IS. Therefore, we examined whether Trp and L-NAT were involved in muscle atrophy in the same manner as IS by performing culture experiments using a human myotube cell line.
RESULTS: The removal efficiency of L-NAT was the same as that of Trp. However, L-NAT concentrations in the pooled sera increased at the end of the experiment. Trp (1 mM) decreased the area of human myocytes, similar to IS, whereas L-NAT did not.
CONCLUSION: L-NAT is a binding competitor with the ability to remove protein-bound IS while preventing sarcopenia.

Keywords: N-acetyl-l-tryptophan; indoxyl sulfate; myotube; uremic sarcopenia

DOI: https://doi.org/10.1111/1744-9987.14047