Redox Regulation of Protein Kinase B/Akt Function by an Allosteric Disulphide Bond

Most proteins in nature are chemically modified after they are made to control how, when and where they function. One type of chemical modification is the cleavage of disulphide bonds that link pairs of cysteine residues in the polypeptide chain. These cleavable bonds are known as allosteric disulphides. From an analysis of labile disulphide bonds in all protein structures from the Protein Data Bank (PDB), my colleagues and I identified a potential allosteric disulphide in the serine/threonine protein kinase B/Akt; linking cysteine residues 60 and 77 in the N-terminus pleckstrin homology (PH) domain. Akt plays a central role in glucose metabolism, cell survival and angiogenesis and is often hyper-activated in cancer cells. Akt is activated at the plasma membrane via binding to phosphatidylinositol-3,4,5-trisphosphate (PIP3) through its PH domain. Dissociation of Akt from the plasma membrane leads to PH domain-mediated autoinhibition of the kinase by a mechanism that is currently unknown. I hypothesised that the PH domain Cys60–Cys77 disulphide is an allosteric bond that regulates autoinhibition and inactivation of the kinase. To elucidate the role of the Cys60–Cys77 disulphide bond in Akt function, wild-type and reduced (Cys60 and/or Cys77 substituted with Ser) PH domain or full-length Akt mutants were analysed for PIP3 plasma membrane binding, Akt phosphorylation and Akt downstream substrate activation, transformation of fibroblasts, and angiogenesis, survival and development of zebrafish. Ablation of the Cys60–Cys77 disulphide bond did not appreciably affect binding of recombinant PH domain to PIP3, but markedly impaired insulin-stimulated binding of full-length Akt to the plasma membrane of adipocytes. Ablation of the Cys60–Cys77 disulphide bond had mixed effects on insulin-stimulated phosphorylation of Akt in fibroblasts. The Cys60Ser mutant was phosphorylated to the same extent as the wild-type, while the Cys77Ser mutant was poorly phosphorylated. Wild-type but not disulphide mutant Akt induced transformation of fibroblasts, indicating an oncogenic role for oxidised but not reduced Akt. Expression of disulphide mutant Akt in zebrafish increased the induction of angiogenesis and development of embryos but did not affect zebrafish survival. My findings imply that the Cys60–Cys77 disulphide bond in the PH domain of Akt is an allosteric disulphide involved in autoinhibition and functioning of the kinase

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases

Data from: Identification of allosteric disulphides from labile bonds in X-ray structures

Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecules of a protein crystal but absent in others, or present in some structures of a protein but absent in others. We reasoned that the labile nature of these disulfides signifies a propensity for cleavage and so possible allosteric regulation of the protein in which the bond resides. A total of 511 labile disulfide bonds were identified. The labile disulfides are more stressed than the average bond, being characterized by high average torsional strain and stretching of the sulfur–sulfur bond and neighbouring bond angles. This pre-stress likely underpins their susceptibility to cleavage. The coagulation, complement and oxygen-sensing hypoxia inducible factor-1 pathways, which are known or have been suggested to be regulated by allosteric disulfides, are enriched in proteins containing labile disulfides. The identification of labile disulfide bonds will facilitate the study of this post-translational modification

Table S1

Excel spreadsheet of unique disulphides in a culled set of X-ray structures described by G. Wang and R. Dunbrack, Jr. (file pdbaanr)

Table S2

Excel spreadsheet of labile disulphide bonds present in some molecules of a protein crystal but absent in others (same PDB, sheet 1), or present in some structures of a protein but absent in others (different PDB, sheet 2)

Supplementary material from Identification of allosteric disulfides from labile bonds in X-ray structures

Table S3, Figures S1-S

Table S2 from Identification of allosteric disulfides from labile bonds in X-ray structures

Excel spreadsheet of labile disulphide bonds present in some molecules of a protein crystal but absent in others (same PDB, sheet 1), or present in some structures of a protein but absent in others (different PDB, sheet 2)

Table S1 from Identification of allosteric disulfides from labile bonds in X-ray structures

Excel spreadsheet of unique disulphides in a culled set of X-ray structures described by G. Wang and R. Dunbrack, Jr. (file pdbaanr)

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells

OBJECTIVE: The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS: Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS: PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION: Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling

Summary: Automated single-cell dispensing is incompatible with white adipose tissue (WAT) due to lipid-laden adipocytes. Single-nuclei RNA-Seq permits transcriptional profiling of all cells from WAT. Human WAT faces unique technical challenges in isolating nuclei compared to rodent tissue due to greater extra-cellular matrix content and larger lipid droplets. In this protocol, we detail how to isolate nuclei from frozen subcutaneous human WAT for single-nuclei RNA-Seq.For complete information on the generation and use of this protocol, please refer to Whytock et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics