Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

Aldose reductases (ARs) belonging to the aldo‐keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP‐DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l‐threose (138 ± 7 mm−1·s−1) followed by d‐erythrose (30 ± 3 mm−1·s−1). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP–low‐molecular‐mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in‐crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four‐carbon sugar alcohol analog, and the enzyme are representative of a near‐cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. Database Structural data are available in the PDB under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCI and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZCM.

26Global and endothelial loss of PTP-PEST is associated with impaired cardiovascular 27 development and embryonic lethality. Although hypoxia is implicated in vascular 28 morphogenesis and remodelling, its effect on PTP-PEST remains unexplored. Here we 29 report that hypoxia (1% oxygen)increases protein levels and catalytic activity of PTP-30 PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry 31 (LC/MS/MS) revealed that AMP-activated protein kinase alpha subunits (AMPK α 1 and 32 α 2 ) interact with PTP-PEST under normoxia but not in hypoxia. experiments confirmed this observation and determined that AMPK α subunits interact 34 with the catalytic domain of PTP-PEST. Knock-down of PTP-PEST abrogated hypoxia 35 mediated tyrosine dephosphorylation and activation of AMPK (Thr 172 phosphorylation). 36Absence of PTP-PEST also blocked hypoxia-induced autophagy (measured as LC3 37 degradation and puncta formation) which was rescued by AMPK activator, metformin 38 (500µM). Since endothelial autophagy is a pre-requisite for angiogenesis, knock-down 39 of PTP-PEST also attenuated endothelial cell migration and capillary tube formation 40 with autophagy inducer rapamycin (200nM) rescuing these effects. In conclusion, this 41 work identifies for the first time PTP-PEST as a regulator of hypoxia-induced AMPK 42 activation and endothelial autophagy to promote angiogenesis.43 44 45 46 47 48 Physiological hypoxia is a potent agonist for embryonic development and post-natal 49 angiogenesis. Oxygen concentrations ranging from 1% to 5% are observed in the 50 uterine environment between embryonic days 3.5 to 14.5 (E3.5-E14.5) to facilitate 51 development of placenta. Similarly, in response to hypoxia (<2% oxygen), endocardial 52 and vascular endothelial cells mediate formation of foetal heart and vasculature 53 respectively in mice between embryonic days E7.5 to E15 [1;2]. Post-natal 54 angiogenesis as seen in female reproductive tract during menstrual cycle in humans, as 55 well as formation of collateral circulation to over-come coronary artery blocks also 56 depend on hypoxia-induced endothelial signalling. Other than the known classical 57 mediators of angiogenesis, multiple studies in rodents have reported increased activity 58 of cytosolic protein tyrosine phosphatases (PTPs) at the sites of post-natal 59 angiogenesis, including ischemic myocardium and skeletal muscles [3;4]. An increase 60 in the cytosolic PTP activity during hypoxia is also seen in the cerebral cortex of new 61 born piglets [5]. Paradoxically, others have shown that non-selective PTP inhibitor, 62 sodium orthovanadate, enhances VEGFR2 signalling and capillary morphogenesis 63 [3;6]. Although these studies allude to the involvement of different PTPs in hypoxia-64 induced angiogenic signalling, barring the involvement of few, for instance VE-PTP (a 65 receptor PTP), the identity of hypoxia responsive cytosolic angiogenic PTPs remains 66 largely unknown. 67 68 Human genome encodes 38 classical PTPs which are specific for tyr...

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Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a wide diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product D-glucuronic acid, at 2.15. 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.