scholarly journals Structure and steroid isomerase activity of Drosophila glutathione transferase E14 essential for ecdysteroid biosynthesis

FEBS Letters ◽  
2020 ◽  
Vol 594 (7) ◽  
pp. 1187-1195 ◽  
Author(s):  
Jana Škerlová ◽  
Helena Lindström ◽  
Elodie Gonis ◽  
Birgitta Sjödin ◽  
Fabrice Neiers ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Isomerase Activity ◽  
Ecdysteroid Biosynthesis

Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization

2010 ◽  
Vol 431 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Natalia Fedulova ◽  
Françoise Raffalli-Mathieu ◽  
Bengt Mannervik
Keyword(s):  
Double Bond ◽  
Glutathione Transferase ◽  
Biological Species ◽  
Glutathione Transferases ◽  
Special Role ◽  
Primary Role ◽  
The Novel ◽  
Protective Function ◽  
Isomerase Activity

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells

2008 ◽  
Vol 414 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Françoise Raffalli-Mathieu ◽  
Carolina Orre ◽  
Mats Stridsberg ◽  
Maryam Hansson Edalat ◽  
Bengt Mannervik
Keyword(s):  
Double Bond ◽  
Glutathione Transferase ◽  
Steroid Hormone ◽  
Cellular Systems ◽  
Double Bond Isomerization ◽  
Steroid Hormone Biosynthesis ◽  
Isomerase Activity ◽  
Cell Extracts ◽  
Hormone Biosynthesis ◽  
Steroidogenic Cells

hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Δ5–Δ4 double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3β-hydroxysteroids catalysed by 3β-HSD (3β-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3β-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Δ5–Δ4 isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Δ5-androstene-3,17-dione into Δ4-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3β-hydroxysteroid oxidoreductase activity of 3β-HSD. The results indicate that the lower isomerase activity of 3β-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.

scholarly journals Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique

2003 ◽  
Vol 374 (3) ◽  
pp. 731-737 ◽  
Author(s):  
Philip G. BOARD ◽  
Matthew C. TAYLOR ◽  
Marjorie COGGAN ◽  
Michael W. PARKER ◽  
Hoffman B. LANTUM ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Glutathione Transferase ◽  
Salt Bridge ◽  
Side Chain ◽  
Active Site Residues ◽  
Conserved Residues ◽  
Isomerase Activity ◽  
Catalytic Role

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.

Updated understanding of platelets in thrombosis and hemostasis: the roles of integrin PSI domains and their potential as therapeutic targets

2020 ◽  
Vol 20 ◽  
Author(s):  
Daniel Thomas MacKeigan ◽  
Tiffany Ni ◽  
Chuanbin Shen ◽  
Tyler William Stratton ◽  
Wenjing Ma ◽  
...  
Keyword(s):  
Redox Reactions ◽  
Platelet Adhesion ◽  
Adaptive Mechanism ◽  
Dynamic Interactions ◽  
Surface Receptors ◽  
Isomerase Activity ◽  
Pathological Conditions ◽  
Fibrinogen Binding ◽  
Immune Mediated ◽  
Novel Target

: Platelets are small blood cells known primarily for their ability to adhere and aggregate at injured vessels to arrest bleeding. However, when triggered under pathological conditions, the same adaptive mechanism of platelet adhesion and aggregation may cause thrombosis, a primary cause of heart attack and stroke. Over recent decades, research has made considerable progress in uncovering the intricate and dynamic interactions that regulate these processes. Integrins are heterodimeric cell surface receptors expressed on all metazoan cells that facilitate cell adhesion, movement, and signaling, to drive biological and pathological processes such as thrombosis and hemostasis. Recently, our group discovered that the plexinsemaphorin-integrin (PSI) domains of the integrin β subunits exert endogenous thiol isomerase activity derived from their two highly conserved CXXC active site motifs. Given the importance of redox reactions in integrin activation and its location in the knee region, this PSI domain activity may be critically involved in facilitating the interconversions between integrin conformations. Our monoclonal antibodies against the β3 PSI domain inhibited its thiol isomerase activity and proportionally attenuated fibrinogen binding and platelet aggregation. Notably, these antibodies inhibited thrombosis without significantly impairing hemostasis or causing platelet clearance. In this review, we will update mechanisms of thrombosis and hemostasis including platelet versatilities and immune-mediated thrombocytopenia, discuss critical contributions of the newly discovered PSI domain thiol isomerase activity, and its potential as a novel target for anti-thrombotic therapies and beyond.

scholarly journals The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

2021 ◽  
Vol 120 (3) ◽  
pp. 979-991
Author(s):  
Rebekah B. Stuart ◽  
Suzanne Zwaanswijk ◽  
Neil D. MacKintosh ◽  
Boontarikaan Witikornkul ◽  
Peter M. Brophy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Liver Fluke ◽  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Affinity Purification ◽  
Economic Loss ◽  
Differential Abundance ◽  
Parasitic Helminths

AbstractFasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

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