Biology subjects

Sharpe, H. J.

Publications and source records attributed to Sharpe, H. J..

4 recordsLinked to original sources

bioRxiv2026

Mapping subcellular H2O2 dynamics reveals tissue specific redox patterns in Drosophila

Redox signalling regulates development, tissue homeostasis, and organismal health. Hydrogen peroxide (H2O2) is a major signalling form of reactive oxygen species (ROS) that modulates protein activity through oxidation of redox-sensitive cysteines that is reversed by cellular reducing systems. Since H2O2 production, scavenging and reduction are spatially restricted, signalling specificity is strongly influenced by subcellular localisation. However, subcellular H2O2 dynamics in animal tissues remain poorly understood. To address this, we generated and validated Drosophila melanogaster lines expressing the ultrasensitive, ultrafast ratiometric H2O2 biosensor HyPer7 targeted to mitochondria, nucleus, cytosol, or plasma membrane. With its highly conserved metabolic and signalling pathways, tractable lifespan, and powerful genetic toolkit, Drosophila is an ideal model for studying redox biology. These new 'FlyPer' lines enable tissue-specific HyPer7 expression and high-resolution measurement of subcellular, in vivo H2O2 dynamics throughout the lifespan. Using FlyPer, we detected compartment-specific H2O2 dynamics during oxidative stress, ageing, wing disc development and embryogenesis, uncovering unexpected patterns of spatially and temporally regulated oxidation throughout the organism. Together, these findings establish FlyPer as a valuable toolkit for in vivo redox biology and suggest that compartmentalised redox dynamics are a fundamental yet still poorly understood layer of developmental programming.

cell biology↗

bioRxiv2023

Protein tyrosine phosphatase receptor kappa regulates glycolysis and de novo lipogenesis to promote hepatocyte metabolic reprogramming in obesity

Fat accumulation, de novo lipogenesis, and glycolysis are key drivers of hepatocyte reprogramming and the consequent metabolic dysfunction-associated steatotic liver disease (MASLD). Here we report that obesity leads to dysregulated expression of hepatic protein-tyrosine phosphatases (PTPs). PTPRK was found to be increased in steatotic hepatocytes in both humans and mice, and positively correlated with PPAR{gamma}-induced lipogenic signalling. High-fat-fed PTPRK knockout mice displayed reduced weight gain and hepatic fat accumulation. Phosphoproteomic analysis in primary hepatocytes and hepatic metabolomics identified fructose-1,6-bisphosphatase 1 and glycolysis as PTPRK targets in metabolic reprogramming. Silencing PTPRK in hepatoma cell lines resulted in reduced colony-forming ability and PTPRK knockout mice developed smaller tumours after diethylnitrosamine-induced hepatocarcinogenesis. Our study defines a novel role for PTPRK in regulating hepatic glycolysis, lipid metabolism, and tumour development. PTPRK inhibition may provide therapeutic possibilities in obesity-associated liver diseases. HighlightsO_LIHepatic receptor-type PTPs are increased in MASLD C_LIO_LIPTPRK is expressed in hepatocytes and upregulated in obesity C_LIO_LIPTPRK deficiency reduces body fat mass and liver steatosis in diet-induced obesity C_LIO_LIPTPRK regulates hepatic glycolysis and lipogenesis, promoting tumorigenesis C_LI

cancer biology↗

bioRxiv2022

Structural determinants of homophilic adhesion by receptor tyrosine phosphatases

The type IIB receptor protein tyrosine phosphatases (R2B RPTPs) are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of R2B RPTPs form stable, homophilic, trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms homodimers; however, the determinants of homophilic specificity remain unknown. We have solved the X-ray crystal structure of the membrane-distal, N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates inter-domain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid, extended molecules that differ in their overall long-range conformation. Furthermore, we identify one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and show that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggest that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences. SIGNIFICANCE STATEMENTCell-cell contacts are dynamically regulated, in part, by the actions of tyrosine kinases and phosphatases. The R2B family of receptor tyrosine phosphatases combine an adhesive extracellular domain with intracellular catalytic domains that bind and dephosphorylate key cell adhesion and polarity proteins. Previous work demonstrated that the extracellular domains form head-to-tail homodimers but, as the interface was composed of residues conserved across the family, homophilic specificity determinants remained unclear. We have used a range of structural techniques including X-ray crystallography, small angle X-ray scattering and AlphaFold modelling to demonstrate that, despite their similarity, two members of the R2B family possess significant differences in their overall shape. Our results support that a combination of subtle shape and sequence variations may determine homophilic binding.

biochemistry↗

bioRxiv2019

The receptor PTPRU is a redox sensitive pseudophosphatase

The dynamic regulation of protein tyrosine phosphorylation is a critical feature of intercellular communication and is regulated by the actions of kinases and phosphatases. The receptor-linked protein tyrosine phosphatases (RPTPs) are key signaling molecules that possess an extracellular domain and intracellular phosphatase domains. Most human RPTPs have tandem intracellular tyrosine phosphatase domains: a catalytically active membrane proximal (D1) domain; and a membrane distal (D2) inactive \"pseudophosphatase\" domain. The receptor PTPRU plays a role in development, multiple cancers and has been implicated in the dephosphorylation of cell adhesion proteins. However, PTPRU has a non-canonical D1 domain containing several sequence variations in key catalytic loops that suggest it may function using a mechanism distinct from related RPTPs. Here, we demonstrate through biochemical and structural studies that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. We show that PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and determine that this is due to substantial disorder in the substrate-binding pocket as well as rearrangement of the catalytic loop such that the active site cysteine is occluded. We also show that this cysteine can form an intramolecular disulfide bond with a vicinal \"backdoor\" cysteine. Further, we demonstrate that the PTPRU D2 domain can recruit substrates of related RPTPs suggesting that this pseudophosphatase functions by competing with active phosphatases for the binding of substrates involved in cell adhesion. Therefore, PTPRU is a bona-fide pseudophosphatase and its functional role in cell signaling is via a non-catalytic mechanism.\n\nSIGNIFICANCE STATEMENTProtein tyrosine phosphorylation is a key post-translational modification required for cellular communication that is dynamically regulated by the activities of tyrosine kinases and phosphatases. Receptor tyrosine phosphatases (RPTPs) possess an extracellular receptor domain and intracellular phosphatase domains. We show that PTPRU is a non-canonical RPTP devoid of catalytic activity and demonstrate that this is due to multiple structural rearrangements. Despite this, PTPRU retains the capacity to bind the substrates of related phosphatases suggesting that the non-catalytic function of this pseudophosphatase is to compete with active phosphatases for the binding of substrates. Such pseudoenzymes represent an exciting and growing area of research with implications as key regulators of signaling networks.

biochemistry↗