Literature summary for 2.8.2.20 extracted from

Byrne, D.P.; Li, Y.; Ngamlert, P.; Ramakrishnan, K.; Eyers, C.E.; Wells, C.; Drewry, D.H.; Zuercher, W.J.; Berry, N.G.; Fernig, D.G.; Eyers, P.A.
New tools for evaluating protein tyrosine sulfation tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors (2018), Biochem. J., 475, 2435-2455 .

Activating Compound

Activating Compound	Comment	Organism	Structure
vemurafenib	complete inhibition of TPST1 at 0.4 mM, vemurafenib potentially induces partial TPST-1 activation at lower concentrations, inhibiting the activity at higher concentrations	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
gene TPST1, recombinant expression of N-terminally His6-tagged TPST1 (residues Lys43-Leu360) lacking the transmembrane domains in Escherichia coli strain BL21(DE3)pLysS in inclusion bodies	Homo sapiens
gene TPST2, recombinant expression of N-terminally His6-tagged TPST2 (residues Gly43-Leu359) lacking the transmembrane domains in Escherichia coli strain BL21(DE3)pLysS in inclusion bodies	Homo sapiens

General Stability

General Stability	Organism
the purified recombinant enzyme is thermally stabilized by PAPS, kinetics, overview	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism
adenosine 3',5'-bisphosphate	product inhibition	Homo sapiens
ATP	-	Homo sapiens
CoA	-	Homo sapiens
dabrafenib	complete inhibition of TPST1 at 0.4 mM	Homo sapiens
dephospho-CoA	inhibits slightly at 0.1 mM, the inhibition is increased by addition of PAPS	Homo sapiens
GW305074X	-	Homo sapiens
GW406108X	-	Homo sapiens
additional information	target-validated protein kinase inhibitors can be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. Biochemical and small-molecule screening by DSF and TPST enzyme assay of the PKIS chemical comprising 367 largely ATP-competitive kinase inhibitors, covering 31 chemotypes originally designed to inhibit 24 distinct protein kinases. Multiple RAF kinase inhibitors target TPST catalytic activity in vitro. Docking analysis of TPST ligandsTo model the interaction of hit and control TPST1/2 ligands, including rottlerin, suramin, the sorafenib-derivative RAF265, and GW305074X with TPST1. Molecular docking is employed to evaluate potential binding modes of compounds using the crystal structure of TPST1 (PDB ID 5WRI). Like TPST2, TPST1 possesses two adjacent docking sites in the extended catalytic region that accommodate binding of substrates, placing the tyrosine-containing substrate (left site) in proximity to the sulfate group of PAPS; target-validated protein kinase inhibitors can be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. Biochemical and small-molecule screening by DSF and TPST enzyme assay of the PKIS chemical comprising 367 largely ATP-competitive kinase inhibitors, covering 31 chemotypes originally designed to inhibit 24 distinct protein kinases. Multiple RAF kinase inhibitors target TPST catalytic activity in vitro. Ligand screening via dual-pronged approach, employing firstly a rapid TPST1 DSF assay and secondly a TPST1 enzyme assay	Homo sapiens
RAF265	a phase I imidazo-benzimidazole RAF inhibitor, complete inhibition of TPST1 at 0.4 mM	Homo sapiens
rottlerin	-	Homo sapiens
sorafenib	complete inhibition of TPST1 at 0.4 mM	Homo sapiens
suramin	-	Homo sapiens
vemurafenib	complete inhibition of TPST1 at 0.4 mM, vemurafenib potentially induces partial TPST-1 activation at lower concentrations, inhibiting the activity at higher concentrations	Homo sapiens
ZM336372	complete inhibition of TPST1 at 0.4 mM	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
Golgi membrane	-	Homo sapiens	139	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
3'-phosphoadenylyl sulfate + protein tyrosine	Homo sapiens	-	adenosine 3',5'-bisphosphate + protein tyrosine-O-sulfate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	O60507	-	-
Homo sapiens	O60704	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged TPST1 (residues Lys43-Leu360) lacking the transmembrane domain, solubilized and refolded from inclusion bodies after expression in Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography and gel filtration	Homo sapiens
recombinant His6-tagged TPST2 (residues Gly43-Leu359) lacking the transmembrane domain, solubilized and refolded from inclusion bodies after expression in Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography and gel filtration	Homo sapiens

Renatured (Commentary)

Renatured (Comment)	Organism
recombinant His6-tagged TPST1 (residues Lys43-Leu360) lacking the transmembrane domains from Escherichia coli strain BL21(DE3)pLysS inclusion bodies by ultrafiltration, dilution in 100 mM Na-acetate, pH 4.5, 6 M guanidine hydrochloride, and 10 mM DTT, concentrated TPST (about 150 mg) is slowly added to refolding buffer containing 50 mM Tris-HCl, pH 8.5, 500 mM guanidine hydrochloride, 10 mM NaCl, 0.4 mM KCl, 0.1 mM EDTA, 0.14 mM n-dodecyl beta-D-maltoside, 5 mM reduced glutathione, and 2.5 mM oxidised glutathione. The refolding mixture is then incubated for 20 h at 4°C without mixing and precipitated protein is subsequently removed by centrifugation	Homo sapiens
recombinant His6-tagged TPST2 (residues Gly43-Leu359) lacking the transmembrane domains from Escherichia coli strain BL21(DE3)pLysS inclusion bodies by ultrafiltration, dilution in 100 mM Na-acetate, pH 4.5, 6 M guanidine hydrochloride, and 10 mM DTT, concentrated TPST (about 150 mg) is slowly added to refolding buffer containing 50 mM Tris-HCl, pH 8.5, 500 mM guanidine hydrochloride, 10 mM NaCl, 0.4 mM KCl, 0.1 mM EDTA, 0.14 mM n-dodecyl beta-D-maltoside, 5 mM reduced glutathione, and 2.5 mM oxidised glutathione. The refolding mixture is then incubated for 20 h at 4°C without mixing and precipitated protein is subsequently removed by centrifugation	Homo sapiens

Renatured (Comment)

Organism

recombinant His6-tagged TPST1 (residues Lys43-Leu360) lacking the transmembrane domains from Escherichia coli strain BL21(DE3)pLysS inclusion bodies by ultrafiltration, dilution in 100 mM Na-acetate, pH 4.5, 6 M guanidine hydrochloride, and 10 mM DTT, concentrated TPST (about 150 mg) is slowly added to refolding buffer containing 50 mM Tris-HCl, pH 8.5, 500 mM guanidine hydrochloride, 10 mM NaCl, 0.4 mM KCl, 0.1 mM EDTA, 0.14 mM n-dodecyl beta-D-maltoside, 5 mM reduced glutathione, and 2.5 mM oxidised glutathione. The refolding mixture is then incubated for 20 h at 4°C without mixing and precipitated protein is subsequently removed by centrifugation

Homo sapiens

recombinant His6-tagged TPST2 (residues Gly43-Leu359) lacking the transmembrane domains from Escherichia coli strain BL21(DE3)pLysS inclusion bodies by ultrafiltration, dilution in 100 mM Na-acetate, pH 4.5, 6 M guanidine hydrochloride, and 10 mM DTT, concentrated TPST (about 150 mg) is slowly added to refolding buffer containing 50 mM Tris-HCl, pH 8.5, 500 mM guanidine hydrochloride, 10 mM NaCl, 0.4 mM KCl, 0.1 mM EDTA, 0.14 mM n-dodecyl beta-D-maltoside, 5 mM reduced glutathione, and 2.5 mM oxidised glutathione. The refolding mixture is then incubated for 20 h at 4°C without mixing and precipitated protein is subsequently removed by centrifugation

Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3'-phosphoadenylyl sulfate + EDFEDYEFDG	GST-tagged CC4-tide, peptide 5-FAM-EDFEDYEFDG-CONH2, from human complement C4 protein	Homo sapiens	adenosine 3',5'-bisphosphate + EDFEDY(sulfated)EFDG	-	?
3'-phosphoadenylyl sulfate + ERHTRSYDYMEGGD	FGF7-tide, peptide 5-FAM-ERHTRSYDYMEGGD-CONH2, from fibroblast growth factor 7	Homo sapiens	adenosine 3',5'-bisphosphate + ERHTRSYDY(sulfated)MEGGD	-	?
3'-phosphoadenylyl sulfate + protein tyrosine	-	Homo sapiens	adenosine 3',5'-bisphosphate + protein tyrosine-O-sulfate	-	?
3'-phosphoadenylyl sulfate + TEYEYLDYDFLPETE	PSGL1-tide, peptide 5-FAM-TEYEYLDYDFLPETE-CONH2, from P-selectin glycoprotein ligand-1	Homo sapiens	adenosine 3',5'-bisphosphate + TEYEYLDY(sulfated)DFLPETE	-	?
3'-phosphoadenylyl sulfate + TTVTDYYYPDIFSS	CCR8-tide, peptide 5-FAM-TTVTDYYYPDIFSS-CONH2, from C-C motif chemokine receptor 8	Homo sapiens	adenosine 3',5'-bisphosphate + TTVTDY(sulfated)YYPDIFSS	-	?
additional information	a microfluidic assay is used to quantify real-time peptide sulfation by TPST1. No activity with peptide lacking the single acceptor tyrosine residue, 5-FAM-EDFEDFEFDG-CONH2	Homo sapiens	?	-	-
additional information	a microfluidic assay is used to quantify real-time peptide sulfation by TPST2. No activity with peptide lacking the single acceptor tyrosine residue, 5-FAM-EDFEDFEFDG-CONH2	Homo sapiens	?	-	-

Synonyms

Synonyms	Comment	Organism
TPST	-	Homo sapiens
TPST1	-	Homo sapiens
TPST2	-	Homo sapiens
tyrosylprotein sulfotransferase	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
20	-	assay at	Homo sapiens

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	the purified recombinant enzyme is stabilized by PAPS, kinetics, overview	Homo sapiens
39.8	-	Tm of purified recombinant His6-tagged TPST2	Homo sapiens
40.3	-	Tm of purified recombinant His6-tagged TPST1	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Homo sapiens

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.0015	-	pH 7.4, 20°C, recombinant His6-tagged enzyme	Homo sapiens	adenosine 3',5'-bisphosphate
0.0065	-	pH 7.4, 20°C, recombinant His6-tagged enzyme	Homo sapiens	RAF265
0.0873	-	pH 7.4, 20°C, recombinant His6-tagged enzyme	Homo sapiens	CoA
0.1665	-	pH 7.4, 20°C, recombinant His6-tagged enzyme	Homo sapiens	dephospho-CoA
0.3786	-	pH 7.4, 20°C, recombinant His6-tagged enzyme	Homo sapiens	ATP

General Information

General Information	Comment	Organism
malfunction	mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. Multiple RAF kinase inhibitors target TPST catalytic activity in vitro	Homo sapiens